Mannanase Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to mannanase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to mannanase variants exhibiting mannanaseactivity, compositions comprising the mannanase variants,polynucleotides encoding the variants, methods of producing thevariants, and methods of using the variants.

Description of the Related Art

Endo-1,4-mannanases (EC 3.2.1.78) are involved in the random hydrolysisof (1→4)-β-D-mannosidic linkages in mannans, galactomannans,glucomannans and galactoglucomanns (Ademark et al. (1998) J. Biotechnol.63:199-210).

Mannan containing polysaccharides are often a major component of thehemicellulose fraction in woods, both softwood and hardwood.

Essentially unsubstituted linear beta-1,4-mannan is found in fruits ofseveral palm trees, such as palm kernels and coconuts. Unsubstitutedbeta-1,4-mannan which is present e.g. in ivory nuts resembles cellulosein the conformation of the individual polysaccharide chains, and iswater-insoluble. In leguminous seeds, water-soluble galactomannan is themain storage carbohydrate comprising up to 20% of the total dry weight.See Moreira et al., (2008) Appl. Microbiol. Biotechnol. 79:165-178.Galactomannans have a linear beta-1,4-mannan backbone substituted withsingle alpha-1,6-galactose, optionally substituted with acetyl groups.Glucomannans are linear polysaccharides with a backbone ofbeta-1,4-linked mannose and glucose alternating in a more or lessregular manner, the backbone optionally being substituted with galactoseand/or acetyl groups. Mannans, galactomannans, glucomannans andgalactoglucomannans glucomannan backbones with branched galactose)contribute to more than 50% of the softwood hemicellulose. Moreover, thecellulose of many red algae contains a significant amount of mannose.

Mannanases have been identified in several Bacillus organisms, but alsofrom other bacteria, fungi, plants, and animals. See, Araujo A. et al.,(1990) I. App. Bacteriol. 68:253-261; Dutta S. et al., (1997) PlantPhysiol. 113: 155-161; Puchar V. et al, (2004) Biochim. Biophys. Acta1674:239-250. Genes encoding these enzymes from a number of organismshave also been cloned and sequenced, many if not all have beenclassified also as members of glycosyl hydrolase (GH) family 5 or 26,based on their sequences. See, e.g., Bewley D. J., (1997) Planta203:454-459; Halstead J. R. et al., (2000) FEMS Microl. Lett. 192:197-203; Xu B. et al., (2002) Eur. J. Biochem. 269: 1753-1760;Henrissat, B. (1991) Biochem. J. 280:309-316.

Beta-mannanases have been used in commercially applications in, forexample, industries such as the paper and pulp industry, foodstuff andfeed industry, pharmaceutical industry and energy industry. Lee J. T.,et al., (2003) Poult. Sci. 82: 1925-1931; McCutchen M. C., et al.,(1996) Biotechnol. Bioeng. 52:332-339; Suurnakki A., et al., (1997) Adv.Biochem. Eng. Biotechnol, 57:261-287.

Within the household care industry, it has been known to use mannanasesin e.g. laundry detergents. In WO 1999/064619 an alkaline mannanase,which exhibites mannanase activity also in the alkaline pH range whenapplied in cleaning compositions, is disclosed.

In WO 2016/054176 other mannanases exhibiting beta-mannanase activityare disclosed.

However, mannanases with improved stability, in particular when used indetergents, have not been disclosed in the prior art. As can be seenfrom the data herein disclosed, the stability of a wild-type mannanasecan be significantly improved by protein engineering. Viewed from acommercial side, providing a mannanase having an improved stability,will have a great impact for the detergent producing industry.

Thus, it is the object of the present invention to provide mannanasevariants with improved stability compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to an isolated mannanase variant, whereinsaid variant has mannanase activity and comprises a modification at oneposition corresponding to a position selected from the positions 1, 2,3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 30, 32,33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60, 62, 63, 65, 66, 67,68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100,104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131, 132, 133, 135,136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167, 169, 172, 173,174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199, 200, 201, 202,203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229, 230, 234, 235,241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260, 261, 262, 266,267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285, 286, 288, 289,290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 of the polypeptideof SEQ ID NO: 2, wherein each modification is independently asubstitution or a deletion,

wherein said variant has at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the mature polypeptide of SEQ ID NO: 1 or thepolypeptide of SEQ ID NO: 2.

The present invention also relates to a composition comprising a variantas herein disclosed, use of such a composition in a domestic orindustrial cleaning process, an isolated polynucleotide encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing the variants as well asmethods of dishwashing or laundering in automatic machines using acomposition herein disclosed.

Definitions

Before the invention is described in further details, it is to beunderstood that the present variants, compositions and methods are notlimited to particular embodiments described, as such may, of course,differ. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Thus, prior to discussing the invention in further dtail, the followingterms will first be defined. In accordance with the detaileddescription, the following abbreviations and defintions apply. Note thatthe singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“an enzyme” includes a plurality of such enzymes, and reference to “thedosage” includes reference to one or more dosages and equivalentsthereof known to those skilled in the art, and so forth.

Certain ranges are presented herein with numerical values being precededby the term “about”. The term “about” as used herein, is to provideliteral support for the exact number that it precedes, as well as anumber that is near to or approximately the number that the termprecedes. In determining whether a number is near to or approximately aspecifically recited number, the near or approximating unrecited numbermay be a number which, in the context in which it is presented, providesthe substantial equivalent of the specifically recited number. Forexample, in connection with a numerical value, the term “about” refersto a range of −10% to +10% of the numerical value, unless term isotherwise specifically defined in context. In another example, thephrase a “pH value of about 9” refers to pH values of from 8.1 to 9.9,unless the pH value is specifically defined otherwise.

Mannanase: The term “mannanase” or “galactomannase” as used hereinrefers to a mannanase enzyme defined as the officially named mannanendo-1,4-beta-mannosidase and having the alternative names beta-mannanseand endo-1,4-mannase. The mannanase term also means a polypeptide orpolypeptide domain of an enzymes that has the ability to catalyze thecleavage or hydrolysis of (1→4) beta-D-mannosidic linkages of mannans,galactomannans, glucomannans, and galactoglucmannans. Thus, it meansthat the mannanase has mannanase activity (EC 3.2.1.78). For purposes ofthe present invention, mannanase activity is determined according to theprocedure described in the Examples. In one aspect, the variants of thepresent invention have at least 20%, e.g., at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 100% of the mannanase activity of the mature polypeptide of SEQID NO: 1.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Comprising: The term “comprising” as used herein refers to, including,but not limited to, the component(s) or feature(s) after the term“comprising”. The component(s) or feature(s) after the term “comprising”are required or mandatory, but the embodiment, may further include othernon-mandatory or optional component(s) or feature(s).

Consisting of: The term “consisting of” as used herein refers to,including, and limited to, the component(s) or feature(s) after the term“consisting of”. The component(s) or feature(s) after the term“consisting of” are therefore required or mandatory, and no othernon-mandatory or optional component(s) or feature(s) are present in theembodimenta.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” as used herein, refersto a linear or circular DNA molecule that comprises a polynucleotideencoding a variant and is operably linked to control sequences thatprovide for its expression. Such control sequences may include apromotor to affect transcription, an optional operator sequence tocontrol transcription, a sequence encoding suitable ribosome-bindingsites on the mRNA, and sequence which control termination oftranscription and translation. Different cell types may be used withdifferent expression vectors.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has mannanseactivity. In one aspect, a fragment contains at least 250 amino acidresidues (e.g., amino acids 250 to 300 of SEQ ID NO: 2), at least 260amino acid residues (e.g., amino acids 260 to 300 of SEQ ID NO: 2), atleast 270 amino acid residues (e.g., amino acids 270 to 300 of SEQ IDNO: 2), or at least 285 amino acid residues (e.g., amino acids 285 to300 of SEQ ID NO: 2.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication. Host cells useful in thepresent invention are generally prokaryotic or eukaryotic host,including any transformable microorganism in which expression can beachieved. Host cells are transformed or transfected with vectorsconstructed using recombinant DNA techniques. Such transformed hostcells may be capable of one or both of replicating the vectors encodingthe variant of the present invention and expressing the desired peptideproduct.

Improved property: The term “improved property” as used herein, refersto a characteristic associated with a variant that is improved comparedto the parent. Such improved properties include, but are not limited to,catalytic efficiency, catalytic rate, in-detergent stability, chemicalstability, oxidation stability, pH activity, pH stability, specificactivity, stability under storage conditions, substrate binding,substrate cleavage, substrate specificity, substrate stability, surfaceproperties, thermal activity, and thermostability.

In-detergent stability: The term “in-detergent stability” as usedherein, refers to the stability of a mannanase enzyme, being both awild-type, parent or variant, when it has been incubated in a detergent.For the purposes of the present invention, in-detergent stability may bedetermined as shown in the Examples.

Isolated: The term “isolated” as used herein, refers to a substance in aform or environment which does not occur in nature. Non-limitingexamples of isolated substances include (1) any non-naturally occurringsubstance, (2) any substance including, but not limited to, any enzyme,variant, nucleic acid, protein, peptide or cofactor, that is at leastpartially removed from one or more or all of the naturally occurringconstituents with which it is associated in nature; (3) any substancemodified by the hand of man relative to that substance found in nature;or (4) any substance modified by increasing the amount of the substancerelative to other components with which it is naturally associated(e.g., multiple copies of a gene encoding the substance; use of astronger promoter than the promoter naturally associated with the geneencoding the substance). An isolated substance may be present in afermentation broth sample.

Mature polypeptide: The term “mature polypeptide” as used herein, refersto a polypeptide in its final form following translation and anypost-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc. In oneaspect, the mature polypeptide is amino acids 1 to 298 of SEQ ID NO: 1based on the SignalP 3.0 predictions (Using neural networks (NN) andhidden Markov models (HMM) trained on Gram-positive bacteria) thatpredicts amino acids −28 to −1 of SEQ ID NO: 1 are a signal peptide. Itis known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” as used herein, refers to a polynucleotide that encodes amature polypeptide having mannanase activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 218 to 1111 of SEQ ID NO: 3.

Mutant: The term “mutant” as used herein, refers to a polynucleotideencoding a variant.

Nucleic acid construct: The term “nucleic acid construct” as usedherein, refers to a nucleic acid molecule, either single- ordouble-stranded, which is isolated from a naturally occurring gene or ismodified to contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic, which comprises one ormore control sequences.

Operably linked: The term “operably linked” as used herein, refers to aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of a polynucleotide such thatthe control sequence directs expression of the coding sequence. Thus,“operably linked” means that a regulatory region o functional domainhaving a known or desired activity, such as a promoter, terminator,signal sequence or enhancer region, is attached to or linked to a target(e.g., a gene or polypeptide) in such a manner as to allow theregulatory region or functional domain to control the expression,secretion or function of that target according to its known or desiredactivity.

Parent or parent mannanase: The term “parent”, “parent mannanse” or“parent polypeptide” as used herein refers to any polypeptide withmannanase activity to which an modification is made to produce theenzyme variants of the present invention. In the present invention, itis to be understood, that a parent polypeptide refers to anaturally-occurring polypeptide that does not include a man-madesubstitution, insertion, or deletion at one or more amino acidpositions. Similarly, the term “parent” with respect to apolynucleotide, refers to a naturally-occurring polynucleotide that doesnot include a man-made nucleoside change. However, a polynucleotideencoding a parent polypeptide is not limited to a naturally-occurringpolynucleotide, but rather encompasses any polynucleotide encoding theparent polypeptide. The parent mannanase may be any mannanase having atleast 60%, such as at least 62%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%sequence identity to SEQ ID NO: 1 or 2.

Polypeptide or enzyme: The terms “polypeptide” and “enzyme” may be usedinterchangeably to refer to polymers of any length comprising amino acidresidues linked by peptide bonds. The conventional one-letter orthree-letter codes for amino acid residues are used herein. The polymermay be linear or branched, it may comprise modified amino acids, and itmay be interrupted by non-amino acids. The terms also encompass an aminoacid polymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.)as well as other modifcations known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”. As used herein, “percent (%) sequence identity” with respectto the amino acid or nucleotide sequence identified herein is defined asthe percentage of amino acid residues or nucleotides in a candidatesequence that are identical with the amino acid residues or nucleotidesin a mannanase sequence as set out in the mature sequence of SEQ ID NO:1 or the sequence of SEQ ID NO: 2, or for nucleotides the sequence ofSEQ ID NO: 3, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Variant: The term “variant” as used herein, refers to a polypeptidehaving mannanase activity comprising an modification, i.e., asubstitution or deletion, at one position. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; and a deletion means removal of the amino acid occupying aposition. The variants of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the mannanase activity ofthe mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2.

Wild-type mannanase: The term “wild-type mannanase” as used herein,refers to a mannanase expressed by a naturally occurring microorganism,such as a bacterium, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another mannanase. The amino acid sequence of anothermannanase is aligned with the mature polypeptide disclosed in SEQ ID NO:2, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 2 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anothermannanase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions: For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions: For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions: For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple modifications: Variants comprising multiple modifications areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different modifications: Where different modifications can be introducedat a position, the different modifications are separated by a comma,e.g., “Arg170Tyr,Glu” represents a substitution of arginine at position170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated mannanase variants which havemannanase activity and comprises a modification at one positioncorresponding to a position selected from the positions 1, 2, 3, 4, 5,6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35,37, 38, 39, 41, 44, 45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71,74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108,110, 111, 114, 115, 116, 118, 119, 131, 132, 133, 135, 136, 139, 142,143, 146, 147, 150, 152, 154, 164, 167, 169, 172, 173, 174, 175, 176,177, 180, 181, 183, 184, 185, 196, 199, 200, 201, 202, 203, 205, 206,210, 212, 213, 214, 215, 226, 228, 229, 230, 234, 235, 241, 242, 243,244, 245, 250, 254, 257, 258, 259, 260, 261, 262, 266, 267, 268, 270,271, 272, 273, 276, 279, 280, 283, 285, 286, 288, 289, 290, 292, 293,294, 295, 296, 298, 299, 300, and 301 of the polypeptide of SEQ ID NO:2, wherein each modification is independently a substitution or adeletion,

wherein said variant has at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the mature polypeptide of SEQ ID NO: 1 or thepolypeptide of SEQ ID NO: 2.

The inventors of the present invention has found that a modification inone of the above listed positions provides a mannanase variants whichhave an improved stability compared to the parent mannanase, i.e. amannanase not comprising a modification in any one of the listedpositions. In particular, the stability may be observed as stability indetergent compositions.

The stability in detergent compositions may herein be referred to a “indetergent stability” and falls under the definition elsewhere describedherein. The terms may be used interchangeably, but constitute the samemeaning and purpose for the present invention. The stability has beendetermined as described in the Examples.

In one embodiment, the alteration is a substitution.

In an embodiment, the alteration is a substitution, wherein saidsubstitution of the naturally-occurring amino acid residue at the oneposition for a different amino acid residue produces a mannanase varianthaving an Improvement Factor of >1.0 for a measure of stability.

When the Improvement Factor (IF) is more than 1.0, it means that thevariant tested has an improved property, such as improved stability,compared to the parent mannanase.

The substitution may be made in any one of the positions correspondingto positions: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60,62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131,132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167,169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199,200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229,230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260,261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285,286, 288, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, and 301,wherein numbering is according to SEQ ID NO: 2.

In another embodiment, the alteration is a deletion.

In another embodiment, the alteration is a deletion, wherein saiddeletion of the naturally-occurring amino acid residue at the oneposition produces a mannanase variant having an Improvement Factorof >1.0 for a measure of stability.

In one embodiment, the deletion is in the position corresponding toposition 201 or 230 wherein numbering is according to SEQ ID NO: 2.

In one embodiment, the improved stability is in-detergent stability orthermostability.

Unless specifically disclosed herein, the numbering of amino acidresidues or positions, are done according to SEQ ID NO: 2.

In one embodiment, the variant comprises one of the followingmodifications compared to the parent mannanase: A1G, A1V, A1I, A1M, A1W,A1S, A1T, A1C, A1Y, A1N, A1D, A1E, A1K, A1R, A1H, A1Q, A1F, N2G, N2R,N2E, N2W, N2V, N2L, N2Q, N2M, N2F, N2A, N2H, N2C, N2Y, S3P, S3V, S3M,S3K, S3W, S3C, S3E, S3Q, S3Y, S3H, S3D, G4D, F5H, F5N, Y6M, Y6H, Y6W,Y6M, Y6F, Y6S, Y6V, Y6K, Y6L, Y6A, Y6R, S8T, S8V, S8E, S8K, S8L, S8A,S8N, S8I, S8F, S8H, S8D, S8W, S8P, S8R, S8Q, S8G, G9E, G9H, G9S, G9K,T10V, T11R, T11D, T11E, T11K, Y13H, Y13F, Y13L, D14S, D14G, D14R, D14W,D14A, D14Q, D14T, D14K, D14N, A15R, N16G, N16I, N16Y, N16V, N16P, N16F,N16K, N16S, G17R, G17N, G17M, G17H, N18R, N18C, N18V, N18K, N18L, N18F,N18T, P19Q, P19N, P19I, P19D, V21T, R23I, A30T, Y32F, Y32W, K33Q, K33P,D34G, D34A, D34E, D34S, D34T, D34I, Q35V, Q35L, Q35K, Q35H, Q35M, Q35C,T37P, T37R, T37A, T37F, T37V, T37Q, T37E, T37Y, T37C, T37H, T37L, T37S,T37N, T38I, T38V, A39I, E41V, E41A, E41C, E41R, E41T, E41K, E41Y, E41F,E41S, E41W, E41P, E41N, E41D, E41L, E45K, A44G, N45A, N45S, N45G, N45P,N45Q, N45T, N45R, N45F, G47A, G47S, G47H, G47M, G47N, G47W, G47L, G47R,G47K, G47Y, G47T, G47F, G47Q, G47C, G47S, G47D, D57N, G59Q, G59S, G59T,G59E, G59W, G59F, Q60R, Q60K, Q60N, Q60C, Q60M, T62D, T62E, T62I, T62Q,T62L, T62M, T62V, T62K, T62Y, T62F, T62H, K63R, K63Q, K63L, D65C, D65E,Q66H, Q66R, H67S, H67T, H67S, H67Y, H67A, H67D, H67R, H67N, H67Q, H67F,H67G, T68W, T68R, T68D, R70K, R70W, R70M, R70L, R70T, R70Q, R70G, R7ON,N71T, N71S, N71L, N71P, N71Q, N71K, N71S, N71W, N71A, S74H, S74T, S74D,S74L, S74K, S74R, S74W, E77T, E77N, E77G, E77F, E77Y, D78G, D78A, D78Q,D78S, D78E, D78R, D78V, D78F, D78A, D78W, D78L, D78H, D78K, D78T, K78Y,K78M, K78N, N79R, N79H, N79C, N79K, H80R, H80S, H80K, H80Y, H80F, H80C,H80W, H80D, L81R, L81N, L81M, V82S, V82I, V82R, A83P, A83S, A83T, Y93W,Y93A, Y93R, Y93L, Y93I, Y93C, Y93V, Y93F, Y93Q, Y93T, Y93M, Y93D, Y93K,Y93S, Y93E, Y93H, S95D, S95E, S95T, S95E, S95C, I96P, I96A, I96F, A97R,A97S, A97D, A97K, A97E, A97N, A97M, S98P, S98D, N100F, N100H, N100Y,N100I, N100L, N100D, N100M, N100C, N100K, R101A, D104S, D104G, D104R,D104Q, D104W, D104A, D104V, D104M, D104I, D104H, I107S, I107A, I107R,I107M, I107K, I107V, E108S, E108Q, E108T, R110T, R110S, R110H, R110G,R110K, R110N, R110V, R110A, R110I, R110Y, R110C, S111P, S111A, S111R,S111W, S111Q, S111G, S111K, S111T, S111G, S111W, S111D, S111H, S111Y,S111V, I114Q, I114M, I114W, I114A, I114E, I114G, I114L, I114R, I114T,I114V, G115D, G115E, G115F, G115R, K116R, K116H, D118R, D118K, D118A,D118T, D118H, D118L, D118N, D118Q, D118S, T119R, S131T, W132C, W132R,W132L, W132N, W132F, W132P, E133R, E133T, E133M, E133Y, E133A, E133H,E133K, E133V, E133C, E133L, D135H, D135M, D135P, D135Y, D135N, D135G,D135E, D135Q, D135K, D135R, D135V, D135C, A136P, A136G, A136C, A136K,A136P, D139R, D139V, D139W, D139Q, D139G, D139A, D139L, D139P, D139T,D139C, K142R, K142Q, K142M, K142S, K142V, K142I, Q143L, Q143M, Q143A,Q143R, Q143D, Q143I, Q143K, Q143C, Q143E, Q143N, Q143H, Q143T, Q143V,Q143W, P146D, R147T, R147E, N150M, N150H, N150V, N150R, N150W, N150T,N150S, N150Q, N150L, N150K, N150G, N150D, N150A, N150E, G152M, G152R,G152D, G152H, N154G, N154C, N154R, N154M, W164G, W164N, W164Q, W164F,W164L, W164M, W164S, F167Y, Q169A, Q169D, Q169T, Q169R, Q169K, H172R,H172W, H172F, H172R, D173E, D173C, Y174R, Y174W, Y174F, Y174L, Y174K,Y174M, G175A, R176A, R176Q, R176E, R176C, R176D, R176V, R176L, R176P,R176K, R176Y, R176S, R176M, R176G, R176H, R176I, E177G, E177S, E177Y,E177Q, E177N, E177H, E177T, E177V, E177M, E177C, E177W, N180A, N180V,N180R, N180A, N180C, N180G, N180K, N180E, N180M, N180Y, N180S, N180T,A181R, A181T, A181V, A181I, A181E, A181K, A181H, P183M, P183T, P183G,P183E, P183H, P183F, P183L, P183A, P183V, P183W, P183D, P183S, Q184T,Q184A, Q184R, Q184S, Q184W, Q184E, Q184K, Q184G, Q184H, Q184C, Q184N,Q184E, Q184F, R185D, R185A, R185V, R185G, R185L, R185I, R185S, R185T,R185C, R185E, R185Y, R185K, Y196W, Y196F, G199A, N200T, N200S, A201*,A201E, S202H, S202T, S202V, S202C, S202N, S202E, S202D, S202W, S202K,S202M, S202R, S202Y, S2021, S202F, S202Q, Q203M, Q203L, Q203V, Q203T,Q203A, R205K, R2051, R205V, T206R, T206C, T206L, T206I, T206P, T206D,T206M, R210N, R210C, R210K, R210M, R210Gm R210L, L212F, L212I, N213I,N213V, N213C, N213D, N213E, N213K, Q214E, D215G, D215W, D215C, D215F,D215N, D215L, R226K, T228S, T228M, N229D, N229C, N229H, N229W, N229Y,G230*, E234F, E234H, E234Y, E234W, A235R, A235K, A235T, A235C, A235E,S241C, E242Q, E242H, E242Y, Q243E, Q243K, Q243C, Q243A, Q243Y, Q243H,Q243R, Q243A, Q243T, Q243A, Q243L, R244K, R244V, G245K, A250G, K254Y,K254T, K254H, K254F, K254W, G257W, G257E, G257A, G257F, G257S, G257Y,G257Q, G257D, G257L, G257M, G257H, P258Q, P258V, P258M, P258W, P258F,E259F, E259C, W260L, W260V, W260F, W260T, W260Y, E261C, E261D, E261A,Y262F, S266A, N267C, N267Y, N267Q, N267F, N267E, N267M, N267V, N267K,N267D, N167W, D268N, A270D, A270E, A270P, A270Y, A270M, A270C, A270Q,G271I, N272M, N272K, N272E, N273E, N273H, N273Y, N273K, N273C, N273Q,N273F, N273D, N273S, N273W, N273A, N273V, N273M, A276N, A276T, A276C,A276E, A276P, A276D, A276W, A276Q, N279D, N279E, N279Q, N279D, N279H,N279Q, T280L, T280C, N283H, N283W, N283Y, N283C, N283F, P285D, P285S,P285R, P285K, Y286C, Y286N, Y286W, Y286F, Y286K, L288I, R289D, R289Q,R289L, R289G, R289C, R289E, E290D, E290A, E290Q, S292A, R293T, R293E,R293H, R293S, L294R, L294V, L294P, L294H, L294K, L294I, L294I, S295V,S295P, S295C, S295F, S295I, S295L, S295K, S295Q, S295A, S295M, S295N,S295R, T296S, F298Y, T299P, T299E, T299C, T299S, T299Y, T299H, T299Q,T299M, T299L, T299G, G300S, G300V, G300M, G300A, G300P, G300F, G300L,G300Q, G300D, G301E, G301F, G301H, G301D, G301A, G301W, G301I, G301Q,G301C, G301L, G301V, and G301R, wherein numbering is according to SEQ IDNO: 2.

In an embodiment, the variant has sequence identity of at least 60%,e.g., at least 62, at least 63%, at least 64%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%, tothe amino acid sequence of the parent mannanase.

In another embodiment, the variant has at least 60%, e.g., at least 62,at least 63%, at least 64%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, such as at least 96%, at least97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of SEQ ID NO: 2.

As described above the stability, such as the in-detergent stability orthermostability, may be determined as described in the Examples. Inparticular, the stability may be determined at a pH of 9.0.

Thus, in one embodiment, the variant of the present invention has animproved stability when measured at pH 9.0.

Accordingly, in one embodiment, the variant comprises a substitution ordeletion at one position corresponding to positions 1, 2, 3, 5, 6, 8, 9,14, 15, 18, 23, 33, 34, 35, 37, 38, 39, 41, 45, 47, 59, 60, 62, 63, 65,66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98,100, 104, 107, 108, 111, 114, 116, 118, 119, 133, 136, 139, 142, 143,146, 147, 150, 169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184,185, 199, 200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 228,229, 230, 234, 235, 241, 242, 243, 244, 245, 257, 258, 259, 260, 261,267, 270, 271, 272, 273, 276, 279, 280, 283, 286, 288, 294, 295, 299,300, and 301 of the polypeptide of SEQ ID NO: 2, and wherein saidsubstitution or deletion of the naturally-occurring amino acid residueat the one position produces a mannanase variant having an ImprovementFactor ≥1.0 for a measure of stability at pH 9.0.

In one embodiment, the variant comprises specific modifications such assubstitution or deletion. Thus, in one embodiment, the variant comprisesone modification selected from the group of: A1G, A1V, A1I, A1M, A1W,A1S, A1T, A1C, A1Y, A1N, A1D, A1E, A1K, A1R, A1H, N2G, N2R, N2H, N2C,N2Y, S3P, S3V, S3M, F5H, F5N, Y6M, Y6H, Y6W, Y6S, Y6V, Y6K, Y6L, Y6A,Y6R, S8T, S8V, S8E, S8K, S8L, S8A, S8N, S8Q, S8G, G9E, G9S, G9K, Y13F,Y13L, D14S, D14G, D14R, D14W, D14A, D14Q, D14T, D14K, D14N, A15R, G17H,N18R, N18C, N18V, N18K, R23I, K33P, D34A, D34E, D34S, D34T, D34I, Q35V,Q35L, Q35H, Q35M, Q35C, T37P, T37R, T37A, T37F, T37V, T37Q, T37E, T37Y,T37C, T37H, T37L, T37S, T37N, A39I, E41V, E41A, N45A, N45S, N45G, E45K,G47A, G47S, G47H, G47M, G47N, G47W, G47L, G47R, G47K, G47Y, G47T, G47F,G47Q, G47C, G59S, G59T, G59E, G59W, G59F, Q60N, Q60C, Q60M, T62F, T62H,K63L, D65C, D65E, Q66H, Q66R, H67S, H67T, H67R, H67N, H67Q, H67F, H67G,T68W, T68R, T68D, R70W, R70M, R70G, R70N, N71T, N71S, N71W, N71A, S74H,S74T, S74D, S74L, S74K, S74W, E77T, E77N, E77G, E77F, E77Y, D78G, D78A,D78Q, D78S, D78E, D78R, D78V, D78K, D78T, K78Y, K78M, K78N, N79R, N79H,N79C, N79K, H80R, H80S, H80K, H80W, H80D, L81R, L81N, L81M, V82I, V82R,A83P, A83S, A83T, Y93W, Y93A, Y93R, Y93L, Y93I, Y93C, Y93V, Y93F, Y93Q,Y93T, Y93M, Y93D, Y93E, Y93H, S95D, S95E, S95C, I96P, I96A, I96F, A97R,A97S, A97N, A97M, S98P, N100F, N100H, N100Y, N100D, N100M, N100C, N100K,R101A, D104S, D104G, D104R, D104Q, D104W, D104A, D104V, D104M, D104I,D104H, I107S, I107A, I107R, E108Q, E108T, S111P, S111A, S111R, S111W,S111Q, S111G, S111K, S111H, S111Y, S111V, I114Q, I114M, I114W, I114A,I114E, I114G, I114L, I114R, I114T, I114V, K116R, K116H, D118R, D118K,D118H, D118L, D118N, D118Q, D118S, T119R, E133R, E133C, E133L, A136P,A136G, D139R, D139V, D139W, D139Q, D139G, D139A, D139L, D139C, K142R,K142Q, K142M, K142S, K142V, Q143L, Q143M, Q143A, Q143R, Q143C, Q143E,Q143N, Q143H, Q143T, Q143V, Q143W, P146D, R147E, N150M, N150H, N150V,N150R, N150W, N150T, N150S, N150Q, N150L, N150D, N150A, N150E, Q169A,Q169D, Q169T, Q169R, Q169K, H172W, H172F, H172R, D173E, D173C, Y174R,Y174L, Y174M, G175A, R176H, R176I, E177G, E177S, E177Y, E177M, E177C,E177W, N180A, N180V, N180R, N180E, N180M, N180Y, N180S, N180T, A181V,A181I, A181E, A181K, A181H, P183M, P183T, P183G, P183D, P183S, Q184T,Q184A, Q184R, Q184S, Q184W, Q184E, Q184K, Q184G, Q184F, R185D, R185A,R185V, R185G, R185L, R185C, R185E, R185Y, R185K, G199A, N200S, A201*,A201E, S202H, S202T, S202V, S202C, S202N, S202E, S202D, S202W, S202I,S202F, S202Q, Q203M, Q203L, Q203A, R205K, R205I, R205V, T206R, T206C,T206I, T206P, T206D, T206M, R210N, R210C, R210G, R210L, R210K, R210M,L212F, N213I, N213V, N213E, N213C, N213D, Q214E, D215F, D215N, D215L,T228M, N229Y, G230*, E234F, E234H, E234Y, E234W, A235R, A235K, A235E,S241C, E242Q, E242H, E242Y, Q243E, Q243K, Q243C, Q243A, Q243Y, Q243H,R244K, G245K, G257W, G257E, G257A, G257F, G257S, G257Y, G257L, G257H,P258Q, P258V, P258M, P258W, P258F, E259F, E259C, W260L, W260V, W260F,W260Y, W260T, E261C, E261D, E261A, N267K, N267D, N167W, A270Y, A270M,A270C, A270Q, G271I, N272K, N272E, N273E, N273D, N273H, N273F, N273W,N273A, N273V, N273M, A276N, A276T, A276W, A276D, A276C, A276Q, N279D,N279E, T280L, T280C, N283H, N283W, N283Y, N283F, Y286C, Y286K, L288I,L294R, L294V, L294P, L294H, L294K, L294I, L294I, S295C, S295F, S295I,S295L, S295K, S295Q, S295A, S295M, S295N, S295R, T299P T299E, T299C,T299S, T299Y, T299H, T299Q, T299M, T299L, T299G, G300S, G300V, G300M,G300A, G300P, G300F, G300L, G300Q, G300D, G301E, G301F, G301H, G301D,G301A, G301W, G301I, G301Q, G301C, G301L, and G301V.

Stability may not only be measured at pH 9.0 but may also be measured ateither more alcaline or acidic pH. The pH that stability is measured atmay vary depending on the detergent composition the variant (or enzyme)needs to be stable in. Thus, the present invention also encompassstability at other pH, such as pH 10.8. Thus, in one embodiment, thevariant of the present invention has an improved stability when measuredat pH 10.8.

Accordingly, in one embodiment, the variant comprises a substitution ordeletion at one position corresponding to positions 1, 2, 3, 8, 9, 10,13, 16, 17, 18, 19, 21, 35, 37, 38, 41, 44, 45, 47, 59, 60, 62, 67, 70,71, 74, 77, 78, 80, 93, 95, 97, 100, 107, 110, 111, 115, 118, 132, 133,135, 136, 139, 142, 143, 147, 150, 152, 154, 164, 167, 169, 174, 176,177, 180, 181, 183, 184, 185, 200, 202, 203, 205, 206, 210, 212, 213,215, 226, 229, 235, 243, 244, 254, 257, 260, 267, 270, 273, 276, 279,283, 285, 286, 289, 290, 292, 293, 294, 296, and 301 of the polypeptideof SEQ ID NO: 2, and wherein said substitution or deletion of thenaturally-occurring amino acid residue at the one position produces amannanase variant having an Improvement Factor ≥1.0 for a measure ofstability at pH 10.8.

In one embodiment, the variant comprises specific modifications such assubstitution or deletion. Thus, in one embodiment, the variant comprisesone modification selected from the group of: A1Y, A1T, A1M, A1K, A1Q,A1F, N2E, N2G, N2W, N2V, N2L, N2Q, N2M, N2F, N2A, S3K, S3W, S3C, S3E,S3Q, S3M, S3Y, S3H, S3D, S8A, S8I, S8F, S8V, S8K, S8L, S8H, S8D, S8W,G9H, T10V, T11R, T11D, T11E, Y13H, N16G, N16I, N16Y, N16V, N16P, N16F,N16K, N16S, G17R, G17N, G17M, N18L, N18F, N18V, N18T, P19Q, P19N, P19I,P19D, V21T, Q35K, Q35V, T37Q, T38I, T38V, E41A, E41V, E41C, E41R, E41T,E41K, E41Y, E41F, E41S, E41W, E41P, E41N, E41D, E41L, A44G, N45P, N45G,N45Q, N45T, N45R, N45S, N45F, G47M, G47T, G47S, G47D, G59T, Q60R, Q60K,T62D, T62E, T62I, T62Q, T62L, T62M, T62V, T62K, T62Y, H67S, H67Y, H67A,H67D, R70M, R70L, R70T, R70Q, N71T, N71L, N71P, N71Q, N71K, N71S, S74K,S74R, S74D, S74H, E77N, D78E, D78V, D78F, D78A, D78Q, D78S, D78W, D78L,D78H, H80Y, H80R, H80F, H80C, Y93M, Y93L, Y93K, Y93C, Y93F, Y93S, S95T,S95E, A97D, A97K, A97E, N100F, N100I, N100L, I107M, I107K, I107V, R110T,R110S, R110H, R110G, R110K, R110N, R110V, R110A, R110I, R110Y, R110C,S111T, S111G, S111W, S111D, G115D, G115E, G115F, G115R, D118A, D118T,W132C, W132R, W132L, W132N, W132F, W132P, E133T, E133M, E133Y, E133A,E133H, E133K, E133V, D135H, D135M, D135Y, A136C, A136K, A136P, D139L,D139P, D139T, K142I, Q143D, Q143I, Q143K, Q143L, R147T, N150R, N150K,N150H, N150G, N150L, N150S, G152M, G152R, G152D, G152H, N154G, N154C,N154R, N154M, W164G, W164N, W164Q, W164F, W164L, W164M, W164S, F167Y,Q169K, Q169R, Y174K, R176A, R176Q, R176E, R176C, R176D, R176V, R176L,R176P, R176K, R176Y, R176S, R176M, R176G, E177Q, E177N, E177H, E177S,E177T, E177V, N180A, N180C, N180G, N180K, A181R, A181T, P183T, P183G,P183E, P183H, P183F, P183L, P183A, P183V, P183W, Q184H, Q184C, Q184N,Q184E, R185I, R185S, R185T, R185D, N200T, S202K, S202M, S202R, S202V,S202Y, Q203M, Q203V, R205K, T206L, T206C, R210G, R210L, L212I, N213E,N213K, D215G, D215W, D215C, R226K, N229C, N229H, N229W, A235T, A235C,Q243K, Q243R, Q243H, Q243A, Q243T, Q243S, Q243L, R244V, K254T, K254H,K254F, K254W, G257Q, G257D, G257L, G257S, G257M, W260Y, W260F, N267C,N267Y, N267Q, N267F, N267E, N267M, N267V, A270E, A270P, N273Y, N273K,N273C, N273Q, N273F, N273D, N273S, A276T, A276P, A276D, A276W, N279Q,N279E, N279D, N279H, N279Q, N283Y, N283C, N283F, N283H, P285D, P285S,P285R, P285K, Y286N, R289D, R289Q, R289L, R289G, R289C, R289E, E290D,E290Q, S292A, R293T, R293E, R293H, R293S, L294R, L294K, L294V, T296S,and G301R.

In one embodiment, is the improved stability of the variant of thepresent invention at least 1.0 for a measure of stability at pH 9.0and/or pH 10.8.

In one embodiment, the improved stability of the variant of the presentinvention is measured either at pH 9.0 or 10.8, or the improvedstability is seen at both pHs, i.e. pH 9.0 and pH 10.8. Thus in oneembodiment, the variant has an improved stability compared to the parentmannanase, when the stability is measured at pH 9.0 and/or pH 10.8.

In one embodiment, the variant has an Improvement Factor of at least 1.5for a measure of stability at pH 9.0 and/or pH 10.8. More particularly,in one embodiment, the variant comprises a substutiton or deletion atone position corresponding to positions 2, 3, 5, 6, 8, 14, 16, 17, 18,23, 34, 35, 37, 38, 41, 45, 47, 60, 62, 66, 68, 70, 71, 77, 78, 80, 81,82, 83, 93, 95, 97, 98, 100, 107, 110, 116, 132, 133, 135, 136, 143,150, 152, 164, 169, 176, 177, 180, 183, 184, 185, 200, 202, 203, 205,206, 210, 215, 229, 234, 235, 242, 243, 244, 254, 257, 258, 260, 261,267, 273, 276, 279, 283, 288, 290, 294, and 295, wherein saidsubstitution or deletion of the naturally-occurring amino acid residueat the one position produces a mannanase variant having an ImprovementFactor ≥1.5 for a measure of stability at pH 9.0 and/or pH 10.8.

In one embodiment, the variant comprises one modification selected fromthe group of: N2E, S3P, S3H, F5H, F5N, Y6F, S8D, S8W, D14R, D14T, D14K,D14N, N16I, N16V, N16P, N16K, N16S, G17R, N18V, N18F, N18T, R23I, D34E,D34I, Q35V, T37Q, T38I, E41A, E41T, E41F, E41S, E41W, E41P, E41N, E41D,E41L, N45P, N45T, N45R, N45S, N45F, G47A, G47S, G47K, G47Q, G47C, G47M,G47T, Q60C, T62I, T62L, T62V, I66R, T68D, R70Q, N71T, N71S, E77T, E77N,E77Y, D78A, H80R, H80K, H80W, L81R, L81N, L81M, V82I, A83T, Y93A, Y93F,Y93M, Y93L, Y93K, Y93C, Y93S, Y93E, S95E, A97D, A97E, S98P, N100L,N100M, I107S, I107M, I107K, I107V, R110H, R110V, R110A, R110C, K116R,W132P, E133A, E133K, E133V, D135H, D135M, D135Y, D135E, D135K, D135R,D135V, A136P, A136K, Q143W, N150W, N150S, N150K, N150L, N150S, G152M,W164G, W164N, W164M, Q169A, Q169T, Q169K, R176A, R176Q, R176E, R176C,R176V, R176L, R176M, R176G, E177S, E177Q, E177N, E177S, N180R, P183T,P183E, P183A, P183V, P183W, Q184E, Q184H, Q184C, R185G, R185T, N200S,S202C, S202E, S202K, S202M, S202V, S202Y, Q203M, Q203V, R205K, T206C,R210G, D215C, N229C, E234Y, A235T, E242Q, Q243E, Q243A, Q243L, R244V,K254F, K254W, G257D, G257L, G257M, P258Q, P258V, W260F, W260Y, E261C,N267Y, N267Q, N273Y, N273C, N273D, N273S, A276D, A276C, N279E, N283H,L288I, E290D, L294R, L294P, L294K, S295I, S295L, S295K, S295A, S295N,and S295R. A variant comprising any one of these modifications have beenshown to have an Improvement Factor of at least 1.5 for a measure ofstability when measured at pH 9.0 and/or pH 10.8 as described in theExamples.

In one embodiment, the variant comprises a modification in one of thepositions corresponding to positions 3, 5, 6, 14, 18, 23, 34, 41, 47,60, 66, 68, 77, 78, 80, 81, 82, 83, 93, 98, 100, 107, 116, 135, 136,143, 150, 169, 177, 180, 184, 185, 200, 202, 210, 234, 242, 243, 258,260, 261, 276, 279, 283, 288, 294, and 295, and has an ImprovementFactor of at least 1.5 for a measure of stability at pH 9.0. In oneparticular embodiment, the variant comprises one of the followingspecific substitutions S3P, F5H, F5N, Y6F, D14R, D14T, D14K, D14N, N18V,D23I, D34E, D34I, E41A, G47A, G47S, G47K, G47T, G47Q, G47C, Q60C, I66R,T68D, E77Y, E77T, E77N, D78A, H80R, H80K, H80W, L81R, L81N, L81M, V82I,A83T, Y93A, Y93F, Y93E, S98P, N100M, I107S, K116R, D135E, D135K, D135R,D135V, A136P, Q143W, N150W, N150S, Q169A, Q169T, Q169K, E177S, N180R,Q184E, R185G, N200S, S202C, S202E, R210G, E234Y, E242Q, Q243E, P258Q,P258V, W260F, E261C, A276D, A276C, N279E, N283H, L288I, L294R, L294P,L294K, S295I, S295L, S295K, S295A, S295N, and S295R, and has anImprovement Factor of at least 1.5 for at measure of stability at pH9.0.

In one embodiment, the variant comprises a modification in one of thepositions corresponding to positions 2, 3, 8, 16, 17, 18, 35, 37, 38,41, 45, 47, 62, 70, 71, 93, 95, 97, 100, 107, 110, 132, 133, 135, 136,150, 152, 164, 169, 176, 177, 183, 184, 185, 202, 203, 205, 206, 215,229, 235, 243, 244, 254, 257, 260, 267, 273, and 290, and has anImprovement Factor of at least 1.5 for a measure of stability at pH10.8. In one particular embodiment, the variant comprises one of thefollowing substitutions N2E, S3H, S8D, S8W, N16I, N16V, N16P, N16K,N16S, G17R, N18F, N18T, Q35V, T37Q, T38I, E41T, E41F, E41S, E41W, E41P,E41N, E41D, E41L, N45P, N45T, N45R, N45S, N45F, G47M, G47T, G47S, T62I,T62L, T62V, R70Q, N71T, N71S, Y93M, Y93L, Y93K, Y93C, Y93S, S95E, A97D,A97E, N100L, I107M, I107K, I107V, R110H, R110V, R110A, R110C, W132P,E133A, E133K, E133V, D135H, D135M, D135Y, A136K, A136P, N150K, N150L,N150S, G152M, W164G, W164N, W164M, Q169K, R176A, R176Q, R176E, R176C,R176V, R176L, R176M, R176G, E177Q, E177N, E177S, P183T, P183E, P183A,P183V, P183W, Q184H, Q184C, R185T, S202K, S202M, S202V, S202Y, Q203M,Q203V, R205K, T206C, D215C, N229C, A235T, Q243A, Q243L, R244V, K254F,K254W, G257D, G257L, G257M, W260Y, N267Y, N267Q, N273Y, N273C, N273D,N273S, and E290D, and has an Improvement Factor of at least 1.5 for ameasure of stability at pH 10.8.

In an even further embodiment, the Improvement Factor of the variant isat least 2.0 for a measure of stability at pH 9.0 and/or pH 10.8. Inparticular, in one embodiment, the variant comprises a substitution ordeletion at one position corresponding to positions 3, 5, 14, 16, 18,38, 41, 45, 47, 60, 62, 66, 68, 71, 77, 78, 80, 81, 93, 95, 97, 98, 107,110, 133, 135, 150, 164, 169, 176, 177, 183, 184, 185, 202, 205, 215,229, 243, 235, 267, 273, 288, 294, and 295 of the polypeptide of SEQ IDNO: 2, and wherein said substitution or deletion of thenaturally-occurring amino acid residue at the one position produces amannanase variant having an Improvement Factor of ≥2.0 for a measure ofstability at pH 9.0 and/or pH 10.8.

In one particular embodiment, the variant comprises one of the specificsubstitutions S3H, F5H, D14R, D14K, N16I, N16P, N16S, N18F, N18T, T38I,E41P, E41D, N45T, N45S, N45F, G47A, G47S, G47K, G47T, G47Q, G47M, G47T,Q60C, T62I, I66R, T68D, N71T, N71S, E77T, D78A, H80R, H80W, L81R, Y93M,Y93C, Y93S, S95E, A97D, A97E, S98P, I107S, I107M, I107K, R110V, E133A,E133K, E133V, D135M, D135Y, N150S, N150K, W164N, W164M, Q169K, Q169K,R176A, R176C, R176V, R176M, R176G, E177Q, E177N, E177S, P183T, P183A,Q184H, R185T, S202K, S202V, S202Y, R205K, D215C, N229C, E234Y, A235T,N267Y, N267Q, N273C, L288I, L294P, L294K, S295I, S295K, S295N, andS295R, and has an Improvement Factor of at least 2.0 for a measure ofstability at pH 9.0 and/or pH 10.8.

In one embodiment, the variant comprises a modification in one of thepositions selected from 5, 14, 47, 60, 66, 68, 77, 78, 80, 81, 98, 107,150, 169, 234, 288, 294, and 295, and has an Improvement Factor of atleast 2.0 for a measure of stability at pH 9.0 and/or pH 10.8. In aparticular embodiment, the variant comprises one of the specificsubstitutions F5H, D14R, D14K, G47A, G47S, G47K, G47T, G47Q, Q60C, I66R,T68D, E77T, D78A, H80OR, H80W, L81R, S98P, I107S, N150S, Q169K, E234Y,L288I, L294P, L294K, S295I, S295K, S295N, and S295R, and has anImprovement Factor of at least 2.0 for a measure of stability at pH 9.0and/or pH 10.8.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 1 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA1G, A1V, A1I, A1M, A1W, A1S, A1T, A1C, A1Y, A1N, A1D, A1E, A1K, A1R,A1H, A1Q, and A1F.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 2 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN2G, N2R, N2E, N2W, N2V, N2L, N2Q, N2M, N2F, N2H, N2C, N2Y, and N2A.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 3 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS3P, S3V, S3M, S3K, S3W, S3C, S3E, S3Q, S3Y, S3H, and S3D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 4 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is G4D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 5 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofF5H and F5N.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 6 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY6M, Y6F, Y6H, Y6S, Y6V, Y6K, Y6F, Y6L, Y6A, Y6R, and Y6W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 8 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS8T, S8V, S8P, S9R, S8E, S8K, S8L, S8A, S8N, S8I, S8F, S8H, S8D, S8Q,S8G, and S8W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 9 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG9E, G9S, G9K, and G9H.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 10 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is T10V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 11 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT11R, T11D, T11K, and T11E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 13 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY13F, Y13L, and Y13H.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 14 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD14S, D14G, D14R, D14W, D14A, D14Q, D14T, D14K, and D14N.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 15 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is A15R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 16 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN16G, N16I, N16Y, N16V, N16P, N16F, N16K, and N16S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 17 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG17R, G17N, G17H, and G17M.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 18 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN18R, N18C, N18V, N18K, N18L, N18F, and N18T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 19 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofP19Q, P19N, P19I, and P19D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 21 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is V21T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 23 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is R23I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 30 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is A30T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 32 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY32F and Y32W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 33 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofK33P and K33Q.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 34 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD34G, D34A, D34E, D34S, D34T, D34I, and D34G.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 35 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ35V, Q35L, Q35H, Q35M, Q35C, and Q35K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 37 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT37P, T37R, T37A, T37F, T37V, T37Q, T37E, T37C, T37H, T37L, T37S, T37N,and T37Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 38 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT38I and T38V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 39 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is A39I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 41 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE41V, E41A, E41C, E41R, E41T, E41K, E41Y, E41F, E41S, E41W, E41P, E41N,E41D, E41Q, and E41L.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 44 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA44N, A44R, and A44G.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 45 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN45A, N45S, N45G, N45P, N45Q, N45T, N45R, N45K, and N45F.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 47 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG47A, G47S, G47H, G47M, G47N, G47W, G47L, G47R, G47K, G47Y, G47T, G47F,G47Q, G47C, G47S, and G47D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 57 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is D57N.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 59 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG59S, G59Q, G59E, G59W, G59F, and G59T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 60 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ60R, Q60N, Q60C, Q60M, and Q60K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 62 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT62D, T62E, T62I, T62Q, T62L, T62M, T62V, T62K, T62F, T62H, and T62Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 63 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofK63R, K63L, and K63Q.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 65 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD65C and D65E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 66 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofI66H and I66R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 67 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofH67S, H67T, H67S, H67Y, H67A, H67R, H67N, H67Q, H67F, H67G, and H67D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 68 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT68R, T68D, and T68W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 70 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR70W, R70M, R70L, R70K, R70T, R70G, R70N, and R70Q.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 71 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN71T, N71S, N71L, N71P, N71Q, N71K, N71W, N71A, and N71S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 74 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS74H, S74T, S74D, S74L, S74K, S74W, and S74R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 77 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE77T, E77N, E77F, E77Y, and E77G.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 78 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD78G, D78A, D78Q, D78S, D78E, D78R, D78V, D78F, D78A, D78W, D78L, D78K,D78I, D78Y, D78M, D78N, and D78H.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 79 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN79H, N79C, N79K, and N79R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 80 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofH80R, H80S, H80K, H80Y, H80F, H80W, H80D, and H80C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 81 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofL81R, L81N, and L81M.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 82 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofV82I, V82S, and V82R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 83 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA83P, A83S, and A83T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 93 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY93W, Y93A, Y93R, Y93L, Y93I, Y93C, Y93V, Y93F, Y93Q, Y93T, Y93M, Y93D,Y93K, Y93E, Y93H, and Y93S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 95 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS95D, S95E, S95T, S95C, and S95E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 96 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofI96P, I96F, and I96A.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 97 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA97R, A97S, A97D, A97K, A97N, A97V, A97M, and A97E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 98 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS98P and S98D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 100 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN100F, N100H, N100Y, N100I, N100D, N100M, N100C, N100K, and N100L.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 101 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD104S, D104G, D104R, D104Q, D104W, D104A, D104M, D104I, D104H, andD104V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 107 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofI107S, I107A, I107R, I107M, I107K, and I107V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 108 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE108S, E108Q, and E108T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 110 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR110T, R110S, R110H, R110G, R110K, R110N, R110V, R110A, R110I, R110Y,and R110C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 111 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS111P, S111A, S111R, S111W, S111Q, S111G, S111K, S111T, S111G, S111W,S111H, S111Y, S111V, and S111D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 114 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofI114Q, I114M, I114A, I114E, I114G, I114L, I114R, I114T, I114V, andI114W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 115 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG115D, G115E, G115F, G115H, G115T, and G115R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 116 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofK116R, K116C, K116T, K116S, K116N, K116V, and K116H.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 118 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD118R, D118K, D118A, D118H, D118L, D118N, D118Q, D118S, and D118T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 119 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is T119R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 131 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ogS131T and S131T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 132 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofW132C, W132R, W132L, W132N, W132F, and W132P.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 133 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE133R, E133T, E133M, E133Y, E133A, E133H, E133K, E133C, E133L, andE133V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 135 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD135H, D135P, D135M, D135N, D135G, D135E, D135Q, D135K, D135R, D135V,D135C, and D135Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 136 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA136P, A136G, A1360, A136K, and A136P.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 139 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD139R, D139V, D139W, D139Q, D139G, D139A, D139L, D139P, D139C, andD139T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 142 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofK142R, K142Q, K142M, K142S, K142V, and K142I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 143 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ143L, Q143M, Q143A, Q143R, Q143D, Q143I, Q143C, Q143E, Q143N, Q143H,Q143T, Q143V, Q143W, and Q143K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 146 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is P146D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 147 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR147E and R147T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 150 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN150M, N150H, N150V, N150R, N150W, N150T, N150S, N150Q, N150L, N150K,N150D, N150A, N150E, and N150G.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 152 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG152M, G152R, G152D, and G152H.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 154 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN154G, N154C, N154R, and N154M.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 164 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofW164G, W164N, W164Q, W164F, W164L, W164M, and W164S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 167 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is F167Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 169 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ169A, Q169D, Q169T, Q169R, and Q169K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 172 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofH172W, H172F, H172E, and H172R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 173 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD173E and D173C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 174 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY174R, Y174W, Y174F, Y174L, Y174M, Y174F, and Y174K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 175 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is G175A.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 176 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR176A, R176Q, R176E, R176C, R176D, R176V, R176L, R176P, R176K, R176Y,R176S, R176M, R176H, R176I, and R176G.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 177 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE177G, E177S, E177Y, E177Q, E177N, E177H, E177T, E177C, E177M, E177W,and E177V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 180 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN180A, N180V, N180R, N180A, N180C, N180G, N180E, N180M, N180Y, N180S,N180T, and N180K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 181 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA181R, A181V, A181I, A181E, A181K, A181H, and A181T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 183 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofP183M, P183T, P183G, P183E, P183H, P183F, P183L, P183A, P183V, P183D,P183S, and P183W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 184 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ184T, Q184A, Q184R, Q184S, Q184W, Q184E, Q184K, Q184G, Q184H, Q184C,Q184N, Q184F, and Q184E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 185 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR185D, R185A, R185V, R185G, R185L, R185I, R185S, R185C, R185E, R185Y,R185K, and R185T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 196 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY196W, Y196F, and Y196F.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 199 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is G199A.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 200 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN200S and N200T.

In another embodiment, the variant comprises or consists of a deletionin the position corresponding to position 201 of the polypeptide of SEQID NO: 2, or of a polypeptide having at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identity to the polypeptide of SEQID NO: 2 which has mannanase activity, and further the variant hasimproved stability compared to the mannanase of SEQ ID NO: 2.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 201 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is A201E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 202 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS202H, S202T, S202V, S202C, S202N, S202E, S202D, S202W, S202K, S202M,S202R, S202I, S202F, S202Q, and S202Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 203 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ203M, 0203L, Q203T, Q203A, and Q203V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 205 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR205I, R205V, and R205K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 206 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT206R, T206C, T206I, T206P, T206D, T206M, and T206L.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 210 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR210N, R210C, R210K, R210M, R210G, and R210L.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 212 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofL212F and L212I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 213 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN213I, N213V, N213C, N213D, N213E, and N213K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 214 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is Q214E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 215 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofD215G, D215W, D215F, D215N, D215L, and D215C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 226 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is R226K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 228 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT228M and T228S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 229 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN229C, N299D, N229H, N229Y, and N229W.

In another embodiment, the variant comprises or consists of a deletionin the position corresponding to position 230 of the polypeptide of SEQID NO: 2, or of a polypeptide having at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% identity to the polypeptide of SEQID NO: 2 which has mannanase activity, and further the variant hasimproved stability compared to the mannanase of SEQ ID NO: 2.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 234 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE234F, E234H, E234Y, and E234W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 235 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA235R, A235K, A235I, A235E, and A235C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 241 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is S241C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 242 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE242Q, E242Y, and E242H.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 243 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofQ243E, Q243K, Q243C, Q243A, Q243Y, Q243H, Q243R, Q243A, Q243T, Q243S,and Q243L.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 244 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR244K and R244V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 245 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is G245K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 250 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is A250G.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 254 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofK254T, K254Y, K254H, K254F, and K254W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 257 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG257W, G257E, G257A, G257F, G257S, G257Y, G257Q, G257D, G257L, G257H,and G257M.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 258 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofP258Q, P258V, P258M, P258F, and P258W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 259 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE259F and E259C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 260 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofW260L, W260V, W260F, W260T, and W260Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 261 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE261D, E261A, and E261C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 262 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is Y262F.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 266 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is S266A.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 267 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN267C, N267Y, N267Q, N267F, N267E, N267M, N267K, N267D, N267W, andN267V.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 268 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is D268N.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 270 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA270E, A270D, A270Y, A270M, A270C, A270Q, and A270P.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 271 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is G271I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 272 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN272K, N272E, N272M, and N272T.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 273 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN273E, N273H, N273Y, N273K, N273C, N273Q, N273F, N273D, N273W, N273A,N273V, N273M, and N273S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 276 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofA276N, A276T, A276C, A276P, A276D, A276E, A276Q, and A276W.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 279 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN279D, N279E, N279Q, N279D, N279H, and N279Q.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 280 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT280L and T280C.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 283 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofN283H, N283W, N283Y, N283C, and N283F.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 285 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofP285D, P285S, P285R, and P285K.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 286 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofY286C, Y286W, Y286F, Y286K, and Y286N.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 288 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is L288I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 289 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR289D, R289Q, R289L, R289G, R289C, and R289E.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 290 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofE290D, E290A, and E290Q.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 292 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is S292A.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 293 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofR293T, R293E, R293H, and R293S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 294 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofL294R, L294V, L294P, L294H, L294K, L294T, and L294I.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 295 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofS295C, S295F, S295I, S295L, S295K, S295Q, S295A, S295M, S295N, S295V,S295P, and S295R.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 296 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is T296S.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 298 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is F298Y.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 299 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofT299E, T299C, T299S, T299Y, T299H, T299T, T299Q, T299M, T299L, T299G,and T299P.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 300 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG300S, G300V, G300M, G300A, G300P, G300F, G300L, G300Q, and G300D.

In another embodiment, the variant comprises or consists of asubstitution in the position corresponding to position 301 of thepolypeptide of SEQ ID NO: 2, or of a polypeptide having at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to thepolypeptide of SEQ ID NO: 2 which has mannanase activity, and furtherthe variant has improved stability compared to the mannanase of SEQ IDNO: 2. In one embodiment, the substitution is selected from the group ofG301E, G301F, G301H, G301D, G301A, G301W, G301I, G301Q, G301C, G301L,G301V, and G301R.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for mannanase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et a., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

The variants may consist of 250 to 300, e.g., 260 to 300, 270 to 300,285 to 300 amino acids.

In an embodiment, the variant has improved specific activity compared tothe parent enzyme.

In an embodiment, the variant has improved stability under storageconditions compared to the parent enzyme.

In an embodiment, the variant has improved thermal activity compared tothe parent enzyme.

In an embodiment, the variant has improved thermostability compared tothe parent enzyme.

Parent Mannanses

The parent mannanase may be (a) a polypeptide having at least 65%sequence identity to the mature polypeptide of SEQ ID NO: 1; (b) apolypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 3, (ii) the full-length complement of (i); or (c) apolypeptide encoded by a polynucleotide having at least 65% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 3.

In an aspect, the parent mannanase has a sequence identity to the maturepolypeptide of SEQ ID NO: 1 of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which has mannanaseactivity. In one aspect, the amino acid sequence of the parent mannansediffers by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 from the mature polypeptide ofSEQ ID NO: 1.

In an aspect, the parent mannanase has a sequence identity to thepolypeptide of SEQ ID NO: 2 of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which has mannanaseactivity. In one aspect, the amino acid sequence of the parent mannansediffers by up to 20 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 from the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 1.

In another aspect, the parent is a fragment of the polypeptide of SEQ IDNO: 2 containing at least 250 amino acid residues, e.g., at least 270and at least 290 amino acid residues.

In another embodiment, the parent is an allelic variant of thepolypeptide of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:3, or (ii) the full-length complement of (i) or (ii) (Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold SpringHarbor, N.Y.).

The polynucleotide of SEQ ID NO: 3 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 3; (ii) the mature polypeptide coding sequence of SEQID NO: 3; (iii) the full-length complement thereof; or (iv) asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film or any otherdetection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 3. In another aspect, the nucleic acid probe is apolynucleotide that encodes the polypeptide of SEQ ID NO: 1; the maturepolypeptide thereof; or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 3.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 3 of at least 65%, e.g., at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial mannanase. For example, the parent may bea Gram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, or Streptomyces mannanase, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma mannanase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis mannanase.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus mannanase.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans mannanase.

In another aspect, the parent is a Bacillus bogoriensis mannanase, e.g.,the mannanase of SEQ ID NO: 1 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving mannanase activity, comprising: (a) introducing into a parentmannanase a modification, such as a deletion or substitution at oneposition corresponding to positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13,14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44,45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80,81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115,116, 118, 119, 131, 132, 133, 135, 136, 139, 142, 143, 146, 147, 150,152, 154, 164, 167, 169, 172, 173, 174, 175, 176, 177, 180, 181, 183,184, 185, 196, 199, 200, 201, 202, 203, 205, 206, 210, 212, 213, 214,215, 226, 228, 229, 230, 234, 235, 241, 242, 243, 244, 245, 250, 254,257, 258, 259, 260, 261, 262, 266, 267, 268, 270, 271, 272, 273, 276,279, 280, 283, 285, 286, 288, 289, 290, 292, 293, 294, 295, 296, 298,299, 300, and 301 of the mature polypeptide of SEQ ID NO: 1, wherein thevariant has mannanase activity; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention. Thus, in one aspect, the presentinvention relates to a polynucleotide encoding a variant comprising amodification at one position corresponding to a position selected fromthe positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60,62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131,132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167,169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199,200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229,230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260,261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285,286, 288, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 ofthe polypeptide of SEQ ID NO: 2, wherein each modification isindependently a substitution or a deletion,

wherein said variant has at least 65%, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the mature polypeptide of SEQ ID NO: 1 or the polypeptide ofSEQ ID NO: 2.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Thus, in one aspect, the present inventionrelates to a polynucleotide encoding a variant comprising a modificationat one position corresponding to a position selected from the positions1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 30,32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60, 62, 63, 65, 66,67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100,104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131, 132, 133, 135,136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167, 169, 172, 173,174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199, 200, 201, 202,203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229, 230, 234, 235,241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260, 261, 262, 266,267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285, 286, 288, 289,290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 of the polypeptideof SEQ ID NO: 2, wherein each modification is independently asubstitution or a deletion, wherein said variant has at least 65%, e.g.,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the mature polypeptide of SEQ IDNO: 1 or the polypeptide of SEQ ID NO: 2, operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thus, inone aspect, the present invention relates to recombinant expressionvectors comprising a polynucleotide encoding a variant comprising amodification at one position corresponding to a position selected fromthe positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60,62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131,132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167,169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199,200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229,230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260,261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285,286, 288, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 ofthe polypeptide of SEQ ID NO: 2, wherein each modification isindependently a substitution or a deletion, wherein said variant has atleast 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the maturepolypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2, apromoter, and transcriptional and translational stop signals.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. Thus, in one aspect, the presentinvention relates to recombinant host cells, comprising a polynucleotideencoding a variant comprising a modification at one positioncorresponding to a position selected from the positions 1, 2, 3, 4, 5,6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35,37, 38, 39, 41, 44, 45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71,74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108,110, 111, 114, 115, 116, 118, 119, 131, 132, 133, 135, 136, 139, 142,143, 146, 147, 150, 152, 154, 164, 167, 169, 172, 173, 174, 175, 176,177, 180, 181, 183, 184, 185, 196, 199, 200, 201, 202, 203, 205, 206,210, 212, 213, 214, 215, 226, 228, 229, 230, 234, 235, 241, 242, 243,244, 245, 250, 254, 257, 258, 259, 260, 261, 262, 266, 267, 268, 270,271, 272, 273, 276, 279, 280, 283, 285, 286, 288, 289, 290, 292, 293,294, 295, 296, 298, 299, 300, and 301 of the polypeptide of SEQ ID NO:2, wherein each modification is independently a substitution or adeletion, wherein said variant has at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the mature polypeptide of SEQ ID NO:

1 or the polypeptide of SEQ ID NO: 2, operably linked to one or morecontrol sequences that direc the production of a variant comprising amodification at one position corresponding to a position selected fromthe positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60,62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131,132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167,169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199,200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229,230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260,261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285,286, 288, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 ofthe polypeptide of SEQ ID NO: 2, wherein each modification isindependently a substitution or a deletion, wherein said variant has atleast 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the maturepolypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2.

A construct or vector comprising a polynucleotide is introduced into ahost cell so that the construct or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier. The term “host cell” encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The choice of a host cell will to a large extentdepend upon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausfi, Bacillus coagulans,Bacillus firm us, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No.

9, 1980).

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant. Thus, in one aspect, the present invention relates tomethods of producing a variant, comprising the steps of (a) cultivatinga host cell comprising a polynucleotide encoding a variant comprising amodification at one position corresponding to a position selected fromthe positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60,62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131,132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167,169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199,200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229,230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260,261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285,286, 288, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 ofthe polypeptide of SEQ ID NO: 2, wherein each modification isindependently a substitution or a deletion, wherein said variant has atleast 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the maturepolypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2, operablylinked to one or more control sequences that direc the production of avariant under conditions suitable for expression of the variant; and (b)recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants, such as a mannanase enzyme assay as describedin Example 2. These detection methods include, but are not limited to,use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate. For example, an enzyme assay maybe used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Methods or Uses Methods for Improving the Nutritional Value of AnimalFeed

The present invention further relates to a method for improving thenutritional value of an animal feed comprising plant based material,comprising adding to the feed a mannanase variant.

The term improving the nutritional value of an animal feed meansimproving the availability of nutrients in the feed. The nutritionalvalues refers in particular to improving the solubilization anddegradation of the arabinoxylan-containing fraction (e.g., such ashemicellulose) of the feed, thereby leading to increased release ofnutrients from cells in the endosperm that have cell walls composed ofhighly recalcitrant hemicellulose. Consequently, an increased release ofarabinoxylan oligomers indicates a disruption of the cell walls and as aresult the nutritional value of the feed is improved resulting inincreased growth rate and/or weight gain and/or feed conversion (i.e.,the weight of ingested feed relative to weight gain). In addition thearabinoxylan oligomer release may result in improved utilization ofthese components per se either directly or by bacterial fermentation inthe hind gut thereby resulting in a production of short chain fattyacids that may be readily absorbed in the hind and utilised in theenergy metabolism.

Compositions of the Invention

The present invention also relates to compositions comprising apolypeptide variant of the present invention. Accordingly, the presentinvention relates to compositions comprising variant comprising amodification at one position corresponding to a position selected fromthe positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60,62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131,132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167,169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199,200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229,230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260,261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285,286, 288, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, and 301 ofthe polypeptide of SEQ ID NO: 2, wherein each modification isindependently a substitution or a deletion, wherein said variant has atleast 65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the maturepolypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2.Preferably, the compositions are enriched in such a variant. The term“enriched” means that the alpha-amylase activity of the composition hasbeen increased, e.g., with an enrichment factor of 1.1.

The composition may comprise a polypeptide variant as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the composition may comprise multiple enzymatic activities, such as anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or xylanase. The additionalenzyme(s) may be produced, for example, by a microorganism belonging tothe genus Aspergillus, e.g., Aspergillus aculeatus, Aspergillus awamori,Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae;Fusarium, e.g., Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulaturn, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumtrichothecioides, or Fusarium venenatum; Humicola, e.g., Humicolainsolens or Humicola lanuginosa; or Trichoderma, e.g., Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the composition may be in the form of a granulate or amicrogranulate. The polypeptide variant may be stabilized in accordancewith methods known in the art.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, e.g., granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries. Thus, the present invention also relates to a detergentadditive comprising a variant of the invention, optionally in the formof a non-dusting granulate, stabilized liquid, or protected enzyme.Accordingly, the present invention relates to a detergent additivecomprising polypeptide having alpha-amylase activity and which exhibitsan improved wash performance and optionally an improved stabilitycompared to the parent polypeptide, said variant comprises at least onemodification in the amino acid motif QSRX1X2X3NR, wherein X1 is Q, K, orR, X2 is L or F, and X3 is A, N, or Q (SEQ ID NO: 2), corresponding toamino acid positions 169 to 176 of SEQ ID NO: 1, and has at least 75%sequence identity to said parent polypeptide, optionally, wherein thedetergent additive is in the form of a non-dusting granulate, stabilizedliquid, or protected enzyme.

In one aspect, the present invention relates to detergent compositionscomprising a polypeptide variant of the present invention in combinationwith one or more additional cleaning composition components.Accordingly, the present invention relates to a detergent compositioncomprising variant comprising a modification at one positioncorresponding to a position selected from the positions 1, 2, 3, 4, 5,6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35,37, 38, 39, 41, 44, 45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71,74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108,110, 111, 114, 115, 116, 118, 119, 131, 132, 133, 135, 136, 139, 142,143, 146, 147, 150, 152, 154, 164, 167, 169, 172, 173, 174, 175, 176,177, 180, 181, 183, 184, 185, 196, 199, 200, 201, 202, 203, 205, 206,210, 212, 213, 214, 215, 226, 228, 229, 230, 234, 235, 241, 242, 243,244,245, 250, 254, 257, 258, 259, 260, 261, 262, 266, 267, 268, 270,271, 272, 273, 276, 279, 280, 283, 285, 286, 288, 289, 290, 292, 293,294, 295, 296, 298, 299, 300, and 301 of the polypeptide of SEQ ID NO:2, wherein each modification is independently a substitution or adeletion, wherein said variant has at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the mature polypeptide of SEQ ID NO: 1 or thepolypeptide of SEQ ID NO: 2, in combination with one or more additionalcleaning composition component.

The choice of additional components is within the skill of the artisanand includes conventional ingredients, including the exemplarynon-limiting components set forth below.

The choice of components may include, for textile care, such as laundry,the consideration of the type of textile to be cleaned, the type and/ordegree of soiling, the temperature at which cleaning is to take place,and the formulation of the detergent product. Although componentsmentioned below are categorized by general header according to aparticular functionality, this is not to be construed as a limitation,as a component may comprise additional functionalities as will beappreciated by the skilled artisan.

Accordingly, the present invention also relates to a composition whichis a cleaning composition.

A composition according to the present invention may further comprise adetergent component, such as a surfactant, a bleach, a dispersantpolymer such as a sulfonated polymer, a complexing agent, a bleachcatalyst such as a manganese bleach catalyst, a crystal growthinhibitor, and/or fabric hueing agents.

In one embodiment, the composition is a phosphate free composition.

The detergent composition of the invention may for example be directedto an ADW (Automatic Dish Wash) composition comprising an enzyme of thepresent invention in combination with one or more additional ADWcomposition components. The choice of additional components is withinthe skill of the artisan and includes conventional ingredients,including the exemplary non-limiting components set forth below.Accordingly, in one aspect, the invention relates to a manual orautomatic dishwashing detergent composition comprising a variant of theinvention, and optionally a surfactant.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.Accordingly, in one aspect, the present invention relates to a manual orautomatic laundry detergent composition comprising a variant accordingto the invention.

In a specific aspect, the invention provides a detergentconcentrate/additive comprising the polypeptide of the invention. Thedetergent additive, as well as the detergent composition, may compriseone or more other enzymes such as an amylase, protease, a lipase, aperoxidase, another amylolytic enzyme, e.g., alpha-amylase,glucoamylase, maltogenic amylase, CGTase and/or a cellulase, anothermannanase, pectinase, pectine lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likepro-teases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274. Preferred commercially available protease enzymes includeALCALASE®, SAVINASE® (SEQ ID NO: 3), PRIMASE®, DURALASE®, ESPERASE®, andKANNASE® (from Novozymes A/S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®,PURAFECT®, PURAFECT OXP®, FN2®, FN3®, FN4® (Genencor InternationalInc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,1131:253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422). Other examples are lipase variants such as those described inWO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ andLIPOLASE ULTRA™ (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of B. licheniformis,described in more detail in GB 1,296,839. Examples of usefulalpha-amylases are the variants described in WO 94/02597, WO 94/18314,WO 96/23873, and WO 97/43424, especially the variants with substitutionsin one or more of the following positions: 15, 23, 105, 106, 124, 128,133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305,391, 408, and 444. Commercially available alpha-amylases are DURAMYL™,LIQUEZYME™, TERMAMYL™, NATALASE™, FUNGAMYL™ and BAN™ (Novozymes A/S),Preferenz S100, Preferenz S110, Preferenz S1000 (SEQ ID NO: 11),Excellenz S110, Excellenz S1000, Excellenz S2000, RAPIDASE™ andPURASTAR™ (from Genencor International Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, 5,776,757 and WO 89/09259. Especially suitable cellulases arethe alkaline or neutral cellulases having colour care benefits. Examplesof such cellulases are cellulases described in EP 0 495 257, EP 0 531372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulasevariants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat.Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299 .

Commercially available cellulases include CELLUZYME®, and CAREZYME®(Novozymes A/S), CLAZINASE®, and PURADAX HA® (Genencor InternationalInc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.Commercially available peroxidases include GUARDZYME® (Novozymes A/S).

Lechinases/Beta-glucanases: Suitable Lechinases include those ofbacterial or fungal origin. They may be chemically modified or proteinengineered. Examples of useful beta-glucanases include those describedin WO 2015/144824 (Novozymes A/S) and WO 99/06516 (Henkel KGAA).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, e.g., granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238 216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually comprise from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually comprise from about0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid mono-ethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glu cam ides”).

The detergent may comprise 0-65% of a detergent builder or complexingagent such as MGDA, GLDA, zeolite, diphosphate, tripho-sphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetri-aminepen-taacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as sulfonated polymers, polyacrylates,maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acidco-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as bleach catalysts, e.g. Mn-basedor Co-based, tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, e.g., WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent composition may comprise about 0-65% by weight, such asabout 5% to about 50% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein laundry/ADW/hard surface cleaning detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), diethanolamine (DEA, alsoknown as 2,2′-iminodiethan-1-ol), triethanolamine (TEA, also known as2,2′,2″-nitrilotriethan-1-ol), and (carboxymethyl)inulin (CMI), andcombinations thereof.

The detergent may comprise 0-30% by weight, such as about 1% to about20%, of a bleaching system. Any bleaching system known in the art foruse in laundry/ADW/hard surface cleaning detergents may be utilized.Suitable bleaching system components include bleaching catalysts,photobleaches, bleach activators, sources of hydrogen peroxide such assodium percarbonate, sodium perborates and hydrogen peroxide—urea (1:1),preformed peracids and mixtures thereof. Suitable preformed peracidsinclude, but are not limited to, peroxycarboxylic acids and salts,diperoxydicarboxylic acids, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone (R), and mixturesthereof. Non-limiting examples of bleaching systems includeperoxide-based bleaching systems, which may comprise, for example, aninorganic salt, including alkali metal salts such as sodium salts ofperborate (usually mono- or tetra-hydrate), percarbonate, persulfate,perphosphate, persilicate salts, in combination with a peracid-formingbleach activator. The term bleach activator is meant herein as acompound which reacts with hydrogen peroxide to form a peracid viaperhydrolysis. The peracid thus formed constitutes the activated bleach.Suitable bleach activators to be used herein include those belonging tothe class of esters, amides, imides or anhydrides. Suitable examples aretetraacetylethylenediamine (TAED), sodium4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS orDOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO98/17767. A particular family of bleach activators of interest wasdisclosed in EP624154 and particulary preferred in that family is acetyltriethyl citrate (ATC). ATC or a short chain triglyceride like triacetinhas the advantage that it is environmentally friendly Furthermore acetyltriethyl citrate and triacetin have good hydrolytical stability in theproduct upon storage and are efficient bleach activators. Finally ATC ismultifunctional, as the citrate released in the perhydrolysis reactionmay function as a builder. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may alsoinclude a bleach catalyst. In some embodiments the bleach component maybe an organic catalyst selected from the group consisting of organiccatalysts having the following formulae:

(iii) and mixtures thereof;wherein each R¹ is independently a branched alkyl group containing from9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons,preferably each R¹ is independently a branched alkyl group containingfrom 9 to 18 carbons or linear alkyl group containing from 11 to 18carbons, more preferably each R¹ is independently selected from thegroup consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl,2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl,isodecyl, isotridecyl and isopentadecyl. Other exemplary bleachingsystems are described, e.g. in WO2007/087258, WO2007/087244,WO2007/087259, EP1867708 (Vitamin K) and WO2007/087242. Suitablephotobleaches may for example be sulfonated zinc or aluminiumphthalocyanines.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step.

The detergent may comprise 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2%or 0.2-1% of a polymer. Any polymer known in the art for use indetergents may be utilized. The polymer may function as a co-builder asmentioned above, or may provide antiredeposition, fiber protection, soilrelease, dye transfer inhibition, grease cleaning and/or anti-foamingproperties. Some polymers may have more than one of the above-mentionedproperties and/or more than one of the below-mentioned motifs. Exemplarypolymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol)(PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) orpoly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine),carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA,poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers,hydrophobically modified CMC (HM-CMC) and silicones, copolymers ofterephthalic acid and oligomeric glycols, copolymers of poly(ethyleneterephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP,poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO)and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplarypolymers include sulfonated polycarboxylates, polyethylene oxide andpolypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Otherexemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of theabove-mentioned polymers are also contemplated.

The detergent compositions of the present invention may also comprisefabric hueing agents such as dyes or pigments, which when formulated indetergent compositions can deposit onto a fabric when said fabric iscontacted with a wash liquor comprising said detergent compositions andthus altering the tint of said fabric through absorption/reflection ofvisible light.

Fluorescent whitening agents emit at least some visible light. Incontrast, fabric hueing agents alter the tint of a surface as theyabsorb at least a portion of the visible light spectrum. Suitable fabrichueing agents include dyes and dye-clay conjugates, and may also includepigments. Suitable dyes include small molecule dyes and polymeric dyes.Suitable small molecule dyes include small molecule dyes selected fromthe group consisting of dyes falling into the Colour Index (C.I.)classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue,Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, ormixtures thereof, for example as described in WO2005/03274,WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated byreference). The detergent composition preferably comprises from about0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt%, or even from about 0.0001 wt % to about 0.04 wt % fabric hueingagent. The composition may comprise from 0.0001 wt % to 0.2 wt % fabrichueing agent, this may be especially preferred when the composition isin the form of a unit dose pouch. Suitable hueing agents are alsodisclosed in, e.g. WO 2007/087257 and WO2007/087243.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the alpha amylase polypeptides of the invention,may be added in an amount corresponding to 0.01-100 mg of enzyme proteinper liter of wash liquor, preferably 0.05-5 mg of enzyme protein perliter of wash liquor, in particular 0.1-1 mg of enzyme protein per literof wash liquor.

The alpha amylase polypeptides of the invention may additionally beincorporated in the detergent formulations disclosed in WO 2006/002643,which is hereby incorporated as reference.

Uses

The present invention is also directed to methods for using apolypeptide variant of the invention. The use may be in detergents, inparticular laundry detergent compositions and dishwashing detergentcompositions. Accordingly, the present invention relates to use of avariant comprising a modification at one position corresponding to aposition selected from the positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13,14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44,45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80,81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115,116, 118, 119, 131, 132, 133, 135, 136, 139, 142, 143, 146, 147, 150,152, 154, 164, 167, 169, 172, 173, 174, 175, 176, 177, 180, 181, 183,184, 185, 196, 199, 200, 201, 202, 203, 205, 206, 210, 212, 213, 214,215, 226, 228, 229, 230, 234, 235, 241, 242, 243, 244, 245, 250, 254,257, 258, 259, 260, 261, 262, 266, 267, 268, 270, 271, 272, 273, 276,279, 280, 283, 285, 286, 288, 289, 290, 292, 293, 294, 295, 296, 298,299, 300, and 301 of the polypeptide of SEQ ID NO: 2, wherein eachmodification is independently a substitution or a deletion, wherein saidvariant has at least 65%, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the mature polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ IDNO: 2.

Thus, the invention provides the use of a polypeptide variant of aparent polypeptide or composition of the invention, in a domestic orindustrial cleaning process. In particular, the invention relates to useof a polypeptide variant according to the invention in laundry,dishwash; such as automatic or manual dishwash, hard surface cleaning,industrial and institutional cleaning, textile desizing, starchmodification, starch liquefaction, saccharification, feed, baking, orbrewing.

In one embodiment, the use is cleaning of fabric, for example laundry.

In another embodiment, the use is cleaning of ceramic, plastic or glassmaterial, for example dishwashing.

Accordingly, the polypeptide variants of the invention are applicable asa component in washing, dishwashing, and hard surface cleaning detergentcompositions (in either a domestic or industrial setting).

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

EXAMPLES Example 1 Site-Saturation Library Generation

The gene of a Mannanase (SEQ ID NO: 3) was cloned into the Bacillussubtilis expression cassette and transformed in a derivative of theexpression host, Bacillus subtilis 168, deficient in alkaline protease,neutral protease, alpha-amylase and pectate lyase. Site-saturationlibraries were generated by the method known as “Mega PCR” approach ineach mentioned position in the Mannanase gene with NNS doping in theforward mutagenic primer. NNS is a well-known method, where the “N”designates any of the four nucleotide bases and “S” designates thenulceotides “C” and “G”.

Two PCR reactions were performed, wherein 1) was generation ofC-terminal fragment with the flanking C-terminal reverse primer and theforward mutagenic primer, and 2) was generation of Mega PCR productusing the C-terminal fragment as the reverse mega-primer and theflanking N-terminal forward primer to give the full-length cassette. TheMega PCR product was then transformed in to the Bacillus host, wheresite-specific homologous recombination in the Bacillus chromosome tookplace.

After 18-20 hours of growth in LB agar media with chloramphinecol forantibiotic selection, the transformed colonies were picked andinoculated in to the aqueous growth media, i.e. TB-Gly media. After 3days of growth, culture PCR was carried out by initial heat lysis ofcells, followed by PCR. The sequence of the PCR products were confirmedby sanger sequencing and the resulting variants were tested in screeningassays. The polymerase used for the PCR reaction was Phusion DNApolymerase (obtained from ThermoScientific, Cat. No.: F530L).

Example 2 Variant Generation by Site-Directed Mutagenesis

The gene of a Mannanase (SEQ ID NO: 3) was cloned into the Bacillussubtilis expression cassette and transformed in the expression host,Bacillus subtilis as described in Example 1. A Mega PCR-basedsite-directed mutagenesis (SDM) was carried out to generate variants ofthe Mannanase gene by introducing mutations at specific sites (asdescribed in Example 1). SDM was carried out using a single mutagenicprimer of 20-30 base pairs with the desired amino acid change(substitution/deletion/insertion) lying in the middle of theoligonucleotide with sufficient flanking residues (9-15 base pairs). TwoPCR reactions were involved 1) generation of C-terminal fragment withthe flanking C-terminal reverse primer and the forward mutagenic primer2) generation of Mega PCR product using the C-terminal fragment as thereverse mega-primer and the flanking N-terminal forward primer to givethe full-length cassette. The Mega PCR product was then transformed into the Bacillus host, where site-specific homologous recombination inthe Bacillus chromosome takes place.

After 18-20 hours of growth in LB agar media with appropriateantibiotic, the transformed colonies were picked and inoculated in tothe aqueous expression media and given for screening assays. The hitsfrom the screening assays were subjected to culture PCR and sent forsequence confirmation. The polymerase used for the PCR reaction wasPhusion DNA polymerase (obtained from ThermoScientific, Cat. No.:F530L).

Example 3 Detergent Stability Determination

The variants generated in Example 1 and 2, were screened for detergentstability at pH 9.0 and pH 10.8. Stability test was performed byincubating the variants in detergent as disclosed in Table 1 (see below)for 16 hrs at 40° C. (pH 9.0) and 3 hrs at 37° C. (pH 10.8) andcomparing the activity of variants with control plate which is incubatedat 4° C. for the same duration.

TABLE 1 Model detergent O Content of compound Compound (% w/w) Na-LAS(linear alkylbenzene sulphonate) 5.3 AEOS (Alkylethoxy sulphate) 10.7Soyfatty acid 1.0 AEO (Alcohol polyethoxylated) 5.3 TEA (Trietanolamine)0.4 Sodium citrate 2.0 CaCl₂ 0.02 Water 75.3 Water hardness adjusted to12°dH by addition of CaCl₂, MgCl₂, and NaHCO₃ (Ca²⁺:Mg²⁺:HCO³⁻ =2:1:4.5) to the test system. After washing the textiles were flushed intap water and dried.

The other detergent used in the Example is commercially availabledetergent Arms & Hammer (A&H) comprising a protease and a proteaseinhibitor besides the detergent ingredients.

TABLE 2 Variants having improved half life Improvement Factor (HIF)compared to the parent mannanase when determined at pH 9.0 Substitutionin parent mannanase Median Temper- Incubation (SEQ ID NO: 2) HIF pHDetergent ature time (hrs) A1G 1.4 9 Model 0 40 16 A1V 1.3 9 Model O 4016 A1I 1.1 9 Model O 40 16 A1M 1.1 9 Model O 40 16 A1W 1.1 9 Model O 4016 A1S 1.1 9 Model O 40 16 A1T 1.1 9 Model O 40 16 A1C 1.1 9 Model O 4016 A1Y 1.2 9 Model O 40 16 A1N 1.0 9 Model O 40 16 A1D 1.1 9 Model O 4016 A1E 1.0 9 Model O 40 16 A1K 1.1 9 Model O 40 16 A1R 1.2 9 Model O 4016 A1H 1.2 9 Model O 40 16 N2G 1.3 9 Model O 40 16 N2R 1.2 9 Model O 4016 S3P 1.9 9 Model O 40 16 S3V 1.1 9 Model O 40 16 S3M 1.2 9 Model O 4016 F5H 2.4 9 Model O 40 16 F5N 1.5 9 Model O 40 16 Y6M 1.2 9 Model O 4016 Y6H 1.2 9 Model O 40 16 Y6W 1.2 9 Model O 40 16 S8T 1.1 9 Model O 4016 S8V 1.2 9 Model O 40 16 S8E 1.2 9 Model O 40 16 S8K 1.2 9 Model O 4016 S8L 1.2 9 Model O 40 16 S8A 1.2 9 Model O 40 16 S8N 1.2 9 Model O 4016 G9E 1.3 9 Model O 40 16 D14S 1.4 9 Model O 40 16 D14G 1.4 9 Model O40 16 D14R 2.6 9 Model O 40 16 D14W 1.2 9 Model O 40 16 D14A 1.4 9 ModelO 40 16 D14Q 1.4 9 Model O 40 16 D14T 1.7 9 Model O 40 16 D14K 2.9 9Model O 40 16 D14N 1.8 9 Model O 40 16 N18R 1.2 9 Model O 40 16 N18C 1.29 Model O 40 16 N18V 1.5 9 Model O 40 16 N18K 1.2 9 Model O 40 16 Q35V1.1 9 Model O 40 16 Q35L 1.1 9 Model O 40 16 T37P 1.4 9 Model O 40 16T37R 1.1 9 Model O 40 16 T37A 1.2 9 Model O 40 16 T37F 1.2 9 Model O 4016 T37V 1.1 9 Model O 40 16 T37Q 1.1 9 Model O 40 16 T37E 1.2 9 Model O40 16 T37Y 1.1 9 Model O 40 16 A39I 1.2 9 Model O 40 16 E41V 1.2 9 ModelO 40 16 E41A 1.6 9 Model O 40 16 N45A 1.0 9 Model O 40 16 N45S 1.1 9Model O 40 16 N45G 1.4 9 Model O 40 16 G47A 2.0 9 Model O 40 16 G47S 2.29 Model O 40 16 G47H 1.3 9 Model O 40 16 G47M 1.3 9 Model O 40 16 G47N1.2 9 Model O 40 16 G47W 1.2 9 Model O 40 16 G47L 1.2 9 Model O 40 16G47R 1.2 9 Model O 40 16 G47K 3.4 9 Model O 40 16 G47Y 1.2 9 Model O 4016 G47T 2.6 9 Model O 40 16 G47F 1.3 9 Model O 40 16 G47Q 2.0 9 Model O40 16 G47C 1.6 9 Model O 40 16 G59S 1.1 9 Model O 40 16 G59T 1.0 9 ModelO 40 16 D65C 1.1 9 Model O 40 16 D65E 1.2 9 Model O 40 16 H67S 1.1 9Model O 40 16 H67T 1.2 9 Model O 40 16 T68W 1.2 9 Model O 40 16 R70W 1.19 Model O 40 16 R70M 1.1 9 Model O 40 16 N71T 1.1 9 Model O 40 16 N71S1.2 9 Model O 40 16 S74H 1.0 9 Model O 40 16 S74T 1.0 9 Model O 40 16S74D 1.2 9 Model O 40 16 S74L 1.1 9 Model O 40 16 S74K 1.2 9 Model O 4016 E77T 2.6 9 Model O 40 16 E77N 1.7 9 Model O 40 16 E77G 1.2 9 Model O40 16 D78G 1.4 9 Model O 40 16 D78A 2.2 9 Model O 40 16 D78Q 1.3 9 ModelO 40 16 D78S 1.4 9 Model O 40 16 D78E 1.4 9 Model O 40 16 D78R 1.4 9Model O 40 16 D78V 1.4 9 Model O 40 16 N79R 1.2 9 Model O 40 16 H80R 2.29 Model O 40 16 H80S 1.2 9 Model O 40 16 H80K 1.6 9 Model O 40 16 L81R2.1 9 Model O 40 16 L81N 1.5 9 Model O 40 16 L81M 1.7 9 Model O 40 16V82I 1.5 9 Model O 40 16 V82R 1.4 9 Model O 40 16 A83P 1.3 9 Model O 4016 A83S 1.2 9 Model O 40 16 A83T 1.5 9 Model O 40 16 Y93W 1.3 9 Model O40 16 Y93A 1.7 9 Model O 40 16 Y93R 1.0 9 Model O 40 16 Y93L 1.2 9 ModelO 40 16 Y93I 1.2 9 Model O 40 16 Y93C 1.2 9 Model O 40 16 Y93V 1.3 9Model O 40 16 Y93F 1.5 9 Model O 40 16 Y93Q 1.2 9 Model O 40 16 Y93T 1.29 Model O 40 16 Y93M 1.2 9 Model O 40 16 Y93D 1.1 9 Model O 40 16 S95D1.2 9 Model O 40 16 S95E 1.2 9 Model O 40 16 I96P 1.2 9 Model O 40 16I96A 1.1 9 Model O 40 16 A97R 1.2 9 Model O 40 16 A97S 1.2 9 Model O 4016 S98P 2.1 9 Model O 40 16 N100F 1.2 9 Model O 40 16 N100H 1.4 9 ModelO 40 16 N100Y 1.2 9 Model O 40 16 R101A 1.2 9 Model O 40 16 D104S 1.2 9Model O 40 16 D104G 1.2 9 Model O 40 16 D104R 1.2 9 Model O 40 16 D104Q1.4 9 Model O 40 16 D104W 1.4 9 Model O 40 16 D104A 1.2 9 Model O 40 16D104V 1.2 9 Model O 40 16 I107S 2.0 9 Model O 40 16 I107A 1.3 9 Model O40 16 I107R 1.4 9 Model O 40 16 S111P 1.0 9 Model O 40 16 S111A 1.2 9Model O 40 16 S111R 1.2 9 Model O 40 16 S111W 1.1 9 Model O 40 16 S111Q1.2 9 Model O 40 16 S111G 1.1 9 Model O 40 16 S111K 1.4 9 Model O 40 16I114Q 1.3 9 Model O 40 16 I114M 1.2 9 Model O 40 16 I114W 1.2 9 Model O40 16 K116R 1.8 9 Model O 40 16 K116H 1.2 9 Model O 40 16 D118R 1.2 9Model O 40 16 D118K 1.3 9 Model O 40 16 TI19R 1.3 9 Model O 40 16 E133R1.4 9 Model O 40 16 A136P 1.5 9 Model O 40 16 A136G 1.2 9 Model O 40 16D139R 1.2 9 Model O 40 16 D139V 1.3 9 Model O 40 16 D139W 1.3 9 Model O40 16 D139Q 1.2 9 Model O 40 16 D139G 1.2 9 Model O 40 16 D139A 1.2 9Model O 40 16 D139L 1.2 9 Model O 40 16 K142R 1.1 9 Model O 40 16 K142Q1.0 9 Model O 40 16 K142M 1.2 9 Model O 40 16 K142S 1.2 9 Model O 40 16K142V 1.2 9 Model O 40 16 Q143L 1.2 9 Model O 40 16 Q143M 1.2 9 Model O40 16 Q143A 1.1 9 Model O 40 16 Q143R 1.2 9 Model O 40 16 N150M 1.2 9Model O 40 16 N150H 1.2 9 Model O 40 16 N150V 1.2 9 Model O 40 16 N150R1.4 9 Model O 40 16 N150W 1.6 9 Model O 40 16 N150T 1.2 9 Model O 40 16N150S 2.6 9 Model O 40 16 N150Q 1.2 9 Model O 40 16 N150L 1.3 9 Model O40 16 Q169A 1.5 9 Model O 40 16 Q169D 1.2 9 Model O 40 16 Q169T 1.6 9Model O 40 16 Q169R 1.4 9 Model O 40 16 Q169K 2.3 9 Model O 40 16 Y174R1.3 9 Model O 40 16 Y174L 1.4 9 Model O 40 16 G175A 1.2 9 Model O 40 16E177G 1.1 9 Model O 40 16 E177S 1.5 9 Model O 40 16 E177Y 1.3 9 Model O40 16 N180A 1.2 9 Model O 40 16 N180V 1.1 9 Model O 40 16 N180R 1.6 9Model O 40 16 P183M 1.2 9 Model O 40 16 P183T 1.2 9 Model O 40 16 P183G1.3 9 Model O 40 16 Q184T 1.2 9 Model O 40 16 Q184A 1.2 9 Model O 40 16Q184R 1.2 9 Model O 40 16 Q184S 1.2 9 Model O 40 16 Q184W 1.4 9 Model O40 16 Q184E 1.6 9 Model O 40 16 Q184K 1.4 9 Model O 40 16 Q184G 1.2 9Model O 40 16 R185D 1.3 9 Model O 40 16 R185A 1.2 9 Model O 40 16 R185V1.2 9 Model O 40 16 R185G 1.5 9 Model O 40 16 R185L 1.2 9 Model O 40 16G199A 1.1 9 Model O 40 16 A201* 1.0 9 Model O 40 16 S202H 1.0 9 Model O40 16 S202T 1.3 9 Model O 40 16 S202V 1.3 9 Model O 40 16 S202C 1.6 9Model O 40 16 S202N 1.1 9 Model O 40 16 S202E 1.7 9 Model O 40 16 S202D1.4 9 Model O 40 16 S202W 1.2 9 Model O 40 16 Q203M 1.2 9 Model O 40 16Q203L 1.2 9 Model O 40 16 R205K 1.2 9 Model O 40 16 T206R 1.2 9 Model O40 16 T206C 1.3 9 Model O 40 16 R210N 1.0 9 Model O 40 16 R210C 1.3 9Model O 40 16 R210G 1.5 9 Model O 40 16 R210L 1.1 9 Model O 40 16 R210K1.2 9 Model O 40 16 R210M 1.4 9 Model O 40 16 L212F 1.2 9 Model O 40 16N213I 1.2 9 Model O 40 16 N213V 1.2 9 Model O 40 16 N213E 1.2 9 Model O40 16 N213C 1.2 9 Model O 40 16 N213D 1.1 9 Model O 40 16 Q214E 1.1 9Model O 40 16 G230* 1.1 9 Model O 40 16 E234F 1.2 9 Model O 40 16 E234H1.2 9 Model O 40 16 E234Y 2.3 9 Model O 40 16 E234W 1.2 9 Model O 40 16A235R 1.4 9 Model O 40 16 A235K 1.3 9 Model O 40 16 S241C 1.1 9 Model O40 16 E242Q 1.6 9 Model O 40 16 E242H 1.2 9 Model O 40 16 Q243E 1.5 9Model O 40 16 Q243K 1.3 9 Model O 40 16 Q243C 1.2 9 Model O 40 16 Q243A1.1 9 Model O 40 16 Q243Y 1.4 9 Model O 40 16 Q243H 1.2 9 Model O 40 16R244K 1.1 9 Model O 40 16 G257W 1.4 9 Model O 40 16 G257E 1.3 9 Model O40 16 G257A 1.4 9 Model O 40 16 G257F 1.4 9 Model O 40 16 G257S 1.3 9Model O 40 16 G257Y 1.2 9 Model O 40 16 G257L 1.4 9 Model O 40 16 P258Q1.7 9 Model O 40 16 P258V 1.9 9 Model O 40 16 P258M 1.1 9 Model O 40 16P258W 1.1 9 Model O 40 16 E259F 1.2 9 Model O 40 16 E259C 1.1 9 Model O40 16 W260L 1.4 9 Model O 40 16 W260V 1.0 9 Model O 40 16 W260F 1.8 9Model O 40 16 W260Y 1.4 9 Model O 40 16 W260T 1.0 9 Model O 40 16 E261C1.6 9 Model O 40 16 N273E 1.3 9 Model O 40 16 N273D 1.2 9 Model O 40 16N273H 1.2 9 Model O 40 16 N273F 1.2 9 Model O 40 16 A276N 1.3 9 Model O40 16 A276T 1.2 9 Model O 40 16 A276W 1.4 9 Model O 40 16 A276D 1.6 9Model O 40 16 A276C 1.6 9 Model O 40 16 N279D 1.3 9 Model O 40 16 N279E1.8 9 Model O 40 16 T280L 1.2 9 Model O 40 16 T280C 1.2 9 Model O 40 16N283H 1.6 9 Model O 40 16 N283W 1.2 9 Model O 40 16 N283Y 1.2 9 Model O40 16 N283F 1.2 9 Model O 40 16 Y286C 1.4 9 Model O 40 16 L288I 3.0 9Model O 40 16 L294R 1.5 9 Model O 40 16 L294V 1.2 9 Model O 40 16 L294P3.5 9 Model O 40 16 L294H 1.2 9 Model O 40 16 L294K 3.5 9 Model O 40 16L294I 1.3 9 Model O 40 16 S295C 1.0 9 Model O 40 16 S295F 1.1 9 Model O40 16 S295I 2.0 9 Model O 40 16 S295L 1.8 9 Model O 40 16 S295K 2.4 9Model O 40 16 S295Q 1.4 9 Model O 40 16 S295A 1.5 9 Model O 40 16 S295M1.3 9 Model O 40 16 S295N 3.3 9 Model O 40 16 S295R 3.0 9 Model O 40 16T299P 1.3 9 Model O 40 16

TABLE 3 Variants having improved half life Improvement Factor (HIF)compared to the parent mannanase when determined at pH 10.8 Substitutionin parent mannanase (SEQ ID Incubation NO: 2) HIF pH DetergentTemperature time (hrs) A1Y 1, 1 10, 8 A & H 37 3 A1T 1, 0 10, 8 A & H 373 A1M 1, 0 10, 8 A & H 37 3 A1K 1, 1 10, 8 A & H 37 3 A1Q 1, 1 10, 8 A &H 37 3 A1F 1, 1 10, 8 A & H 37 3 N2E 1, 6 10, 8 A & H 37 3 N2G 1, 1 10,8 A & H 37 3 N2W 1, 2 10, 8 A & H 37 3 N2V 1, 0 10, 8 A & H 37 3 N2L 1,2 10, 8 A & H 37 3 N2Q 1, 1 10, 8 A & H 37 3 N2M 1, 1 10, 8 A & H 37 3N2F 1, 3 10, 8 A & H 37 3 N2A 1, 1 10, 8 A & H 37 3 S3K 1, 0 10, 8 A & H37 3 S3W 1, 2 10, 8 A & H 37 3 S3C 1, 2 10, 8 A & H 37 3 S3E 1, 1 10, 8A & H 37 3 S3Q 1, 2 10, 8 A & H 37 3 S3M 1, 3 10, 8 A & H 37 3 S3Y 1, 310, 8 A & H 37 3 S3H 2, 2 10, 8 A & H 37 3 S3D 1, 2 10, 8 A & H 37 3 S8A1, 1 10, 8 A & H 37 3 S8I 1, 3 10, 8 A & H 37 3 S8F 1, 4 10, 8 A & H 373 S8V 1, 1 10, 8 A & H 37 3 S8K 1, 2 10, 8 A & H 37 3 S8L 1, 3 10, 8 A &H 37 3 S8H 1, 2 10, 8 A & H 37 3 S8D 1, 6 10, 8 A & H 37 3 S8W 1, 5 10,8 A & H 37 3 G9H 1, 1 10, 8 A & H 37 3 T10V 1, 2 10, 8 A & H 37 3 T11R1, 1 10, 8 A & H 37 3 T11D 1, 1 10, 8 A & H 37 3 T11E 1, 0 10, 8 A & H37 3 Y13H 1, 1 10, 8 A & H 37 3 N16G 1, 4 10, 8 A & H 37 3 N16I 2, 0 10,8 A & H 37 3 N16Y 1, 3 10, 8 A & H 37 3 N16V 1, 6 10, 8 A & H 37 3 N16P2, 2 10, 8 A & H 37 3 N16F 1, 2 10, 8 A & H 37 3 N16K 1, 7 10, 8 A & H37 3 N16S 2, 1 10, 8 A & H 37 3 G17R 1, 5 10, 8 A & H 37 3 G17N 1, 0 10,8 A & H 37 3 G17M 1, 0 10, 8 A & H 37 3 N18L 1, 1 10, 8 A & H 37 3 N18F2, 0 10, 8 A & H 37 3 N18V 1, 0 10, 8 A & H 37 3 N18T 2, 1 10, 8 A & H37 3 P19Q 1, 0 10, 8 A & H 37 3 P19N 1, 1 10, 8 A & H 37 3 P19I 1, 0 10,8 A & H 37 3 P19D 1, 4 10, 8 A & H 37 3 V21T 1, 4 10, 8 A & H 37 3 Q35K1, 3 10, 8 A & H 37 3 Q35V 1, 5 10, 8 A & H 37 3 T37Q 1, 6 10, 8 A & H37 3 T38I 3, 3 10, 8 A & H 37 3 T38V 1, 3 10, 8 A & H 37 3 E41A 1, 2 10,8 A & H 37 3 E41V 1, 2 10, 8 A & H 37 3 E41C 1, 4 10, 8 A & H 37 3 E41R1, 0 10, 8 A & H 37 3 E41T 1, 8 10, 8 A & H 37 3 E41K 1, 0 10, 8 A & H37 3 E41Y 1, 2 10, 8 A & H 37 3 E41F 1, 5 10, 8 A & H 37 3 E41S 1, 9 10,8 A & H 37 3 E41W 1, 5 10, 8 A & H 37 3 E41P 2, 4 10, 8 A & H 37 3 E41N1, 8 10, 8 A & H 37 3 E41D 2, 6 10, 8 A & H 37 3 E41L 1, 6 10, 8 A & H37 3 A44G 1, 2 10, 8 A & H 37 3 N45P 1, 8 10, 8 A & H 37 3 N45G 1, 1 10,8 A & H 37 3 N45Q 1, 0 10, 8 A & H 37 3 N45T 2, 9 10, 8 A & H 37 3 N45R1, 5 10, 8 A & H 37 3 N45S 2, 1 10, 8 A & H 37 3 N45F 2, 0 10, 8 A & H37 3 G47M 2, 0 10, 8 A & H 37 3 G47T 2, 0 10, 8 A & H 37 3 G47S 1, 8 10,8 A & H 37 3 G47D 1, 4 10, 8 A & H 37 3 G59T 1, 2 10, 8 A & H 37 3 Q60R1, 4 10, 8 A & H 37 3 Q60K 1, 4 10, 8 A & H 37 3 T62D 1, 3 10, 8 A & H37 3 T62E 1, 4 10, 8 A & H 37 3 T62I 2, 1 10, 8 A & H 37 3 T62Q 1, 2 10,8 A & H 37 3 T62L 1, 6 10, 8 A & H 37 3 T62M 1, 3 10, 8 A & H 37 3 T62V1, 6 10, 8 A & H 37 3 T62K 1, 2 10, 8 A & H 37 3 T62Y 1, 3 10, 8 A & H37 3 H67S 1, 2 10, 8 A & H 37 3 H67Y 1, 1 10, 8 A & H 37 3 H67A 1, 3 10,8 A & H 37 3 H67D 1, 1 10, 8 A & H 37 3 R70M 1, 1 10, 8 A & H 37 3 R70L1, 0 10, 8 A & H 37 3 R70T 1, 0 10, 8 A & H 37 3 R70Q 1, 7 10, 8 A & H37 3 N71T 2, 2 10, 8 A & H 37 3 N71L 1, 1 10, 8 A & H 37 3 N71P 1, 2 10,8 A & H 37 3 N71Q 1, 4 10, 8 A & H 37 3 N71K 1, 3 10, 8 A & H 37 3 N71S3, 4 10, 8 A & H 37 3 S74K 1, 2 10, 8 A & H 37 3 S74R 1, 2 10, 8 A & H37 3 S74D 1, 0 10, 8 A & H 37 3 S74H 1, 3 10, 8 A & H 37 3 E77N 1, 1 10,8 A & H 37 3 D78E 1, 0 10, 8 A & H 37 3 D78V 1, 0 10, 8 A & H 37 3 D78F1, 1 10, 8 A & H 37 3 D78A 1, 1 10, 8 A & H 37 3 D78Q 1, 2 10, 8 A & H37 3 D78S 1, 3 10, 8 A & H 37 3 D78W 1, 1 10, 8 A & H 37 3 D78L 1, 1 10,8 A & H 37 3 D78H 1, 3 10, 8 A & H 37 3 H80Y 1, 1 10, 8 A & H 37 3 H80R1, 0 10, 8 A & H 37 3 H80F 1, 3 10, 8 A & H 37 3 H80C 1, 1 10, 8 A & H37 3 Y93M 2, 1 10, 8 A & H 37 3 Y93L 1, 5 10, 8 A & H 37 3 Y93K 1, 8 10,8 A & H 37 3 Y930 2, 8 10, 8 A & H 37 3 Y93F 1, 3 10, 8 A & H 37 3 Y93S2, 3 10, 8 A & H 37 3 S95T 1, 4 10, 8 A & H 37 3 S95E 2, 2 10, 8 A & H37 3 A97D 2, 0 10, 8 A & H 37 3 A97K 1, 1 10, 8 A & H 37 3 A97E 2, 0 10,8 A & H 37 3 N100F 1, 3 10, 8 A & H 37 3 N1001 1, 2 10, 8 A & H 37 3N100L 1, 6 10, 8 A & H 37 3 I107M 2, 2 10, 8 A & H 37 3 I107K 2, 9 10, 8A & H 37 3 I107V 1, 7 10, 8 A & H 37 3 R110T 1, 4 10, 8 A & H 37 3 R110S1, 3 10, 8 A & H 37 3 R110H 1, 5 10, 8 A & H 37 3 R110G 1, 3 10, 8 A & H37 3 R110K 1, 2 10, 8 A & H 37 3 R110N 1, 1 10, 8 A & H 37 3 R110V 2, 110, 8 A & H 37 3 R110A 1, 5 10, 8 A & H 37 3 R110I 1, 1 10, 8 A & H 37 3R110Y 1, 3 10, 8 A & H 37 3 R110C 1, 7 10, 8 A & H 37 3 S111T 1, 0 10, 8A & H 37 3 S111G 1, 2 10, 8 A & H 37 3 S111W 1, 4 10, 8 A & H 37 3 S111D1, 0 10, 8 A & H 37 3 G115D 1, 1 10, 8 A & H 37 3 G115E 1, 3 10, 8 A & H37 3 G115F 1, 2 10, 8 A & H 37 3 G115R 1, 0 10, 8 A & H 37 3 D118A 1, 410, 8 A & H 37 3 D118T 1, 1 10, 8 A & H 37 3 W132C 1, 1 10, 8 A & H 37 3W132R 1, 1 10, 8 A & H 37 3 W132L 1, 0 10, 8 A & H 37 3 W132N 1, 2 10, 8A & H 37 3 W132F 1, 3 10, 8 A & H 37 3 W132P 1, 7 10, 8 A & H 37 3 E133T1, 3 10, 8 A & H 37 3 E133M 1, 4 10, 8 A & H 37 3 E133Y 1, 3 10, 8 A & H37 3 E133A 2, 8 10, 8 A & H 37 3 E133H 1, 0 10, 8 A & H 37 3 E133K 2, 110, 8 A & H 37 3 E133V 2, 1 10, 8 A & H 37 3 D135H 1, 8 10, 8 A & H 37 3D135M 3, 3 10, 8 A & H 37 3 D135Y 2, 0 10, 8 A & H 37 3 A136C 1, 1 10, 8A & H 37 3 A136K 1, 5 10, 8 A & H 37 3 A136P 1, 7 10, 8 A & H 37 3 D139L1, 0 10, 8 A & H 37 3 D139P 1, 2 10, 8 A & H 37 3 D139T 1, 1 10, 8 A & H37 3 K142I 1, 1 10, 8 A & H 37 3 0143D 1, 1 10, 8 A & H 37 3 Q143I 1, 310, 8 A & H 37 3 0143K 1, 1 10, 8 A & H 37 3 0143L 1, 3 10, 8 A & H 37 3R147T 1, 2 10, 8 A & H 37 3 N150R 1, 1 10, 8 A & H 37 3 N150K 2, 3 10, 8A & H 37 3 N150H 1, 1 10, 8 A & H 37 3 N150G 1, 3 10, 8 A & H 37 3 N150L1, 7 10, 8 A & H 37 3 N150S 1, 6 10, 8 A & H 37 3 G152M 1, 5 10, 8 A & H37 3 G152R 1, 0 10, 8 A & H 37 3 G152D 1, 1 10, 8 A & H 37 3 G152H 1, 010, 8 A & H 37 3 N154G 1, 1 10, 8 A & H 37 3 N154C 1, 0 10, 8 A & H 37 3N154R 1, 2 10, 8 A & H 37 3 N154M 1, 1 10, 8 A & H 37 3 W164G 1, 6 10, 8A & H 37 3 W164N 2, 3 10, 8 A & H 37 3 W164Q 1, 2 10, 8 A & H 37 3 W164F1, 2 10, 8 A & H 37 3 W164L 1, 1 10, 8 A & H 37 3 W164M 2, 2 10, 8 A & H37 3 W164S 1, 2 10, 8 A & H 37 3 F167Y 1, 2 10, 8 A & H 37 3 Q169K 2, 510, 8 A & H 37 3 Q169R 1, 4 10, 8 A & H 37 3 Y174K 1, 1 10, 8 A & H 37 3R176A 3, 1 10, 8 A & H 37 3 R176Q 1, 7 10, 8 A & H 37 3 R176E 1, 6 10, 8A & H 37 3 R176C 2, 0 10, 8 A & H 37 3 R176D 1, 0 10, 8 A & H 37 3 R176V3, 1 10, 8 A & H 37 3 R176L 1, 7 10, 8 A & H 37 3 R176P 1, 1 10, 8 A & H37 3 R176K 1, 3 10, 8 A & H 37 3 R176Y 1, 1 10, 8 A & H 37 3 R176S 1, 210, 8 A & H 37 3 R176M 2, 9 10, 8 A & H 37 3 R176G 2, 9 10, 8 A & H 37 3E177Q 2, 4 10, 8 A & H 37 3 E177N 2, 0 10, 8 A & H 37 3 E177H 1, 0 10, 8A & H 37 3 E177S 2, 1 10, 8 A & H 37 3 E177T 1, 2 10, 8 A & H 37 3 E177V1, 1 10, 8 A & H 37 3 N180A 1, 2 10, 8 A & H 37 3 N180C 1, 1 10, 8 A & H37 3 N180G 1, 0 10, 8 A & H 37 3 N180K 1, 3 10, 8 A & H 37 3 A181R 1, 010, 8 A & H 37 3 A181T 1, 1 10, 8 A & H 37 3 P183T 2, 6 10, 8 A & H 37 3P183G 1, 1 10, 8 A & H 37 3 P183E 1, 6 10, 8 A & H 37 3 P183H 1, 1 10, 8A & H 37 3 P183F 1, 3 10, 8 A & H 37 3 P183L 1, 1 10, 8 A & H 37 3 P183A2, 1 10, 8 A & H 37 3 P183V 1, 5 10, 8 A & H 37 3 P183W 1, 8 10, 8 A & H37 3 Q184H 2, 3 10, 8 A & H 37 3 Q184C 1, 8 10, 8 A & H 37 3 Q184N 1, 110, 8 A & H 37 3 Q184E 1, 1 10, 8 A & H 37 3 R185I 1, 2 10, 8 A & H 37 3R185S 1, 3 10, 8 A & H 37 3 R185T 2, 0 10, 8 A & H 37 3 R185D 1, 3 10, 8A & H 37 3 N200T 1, 4 10, 8 A & H 37 3 S202K 2, 2 10, 8 A & H 37 3 S202M1, 5 10, 8 A & H 37 3 S202R 1, 1 10, 8 A & H 37 3 S202V 2, 1 10, 8 A & H37 3 S202Y 2, 2 10, 8 A & H 37 3 Q203M 1, 5 10, 8 A & H 37 3 Q203V 1, 610, 8 A & H 37 3 R205K 2, 3 10, 8 A & H 37 3 T206L 1, 3 10, 8 A & H 37 3T206C 1, 8 10, 8 A & H 37 3 R210G 1, 4 10, 8 A & H 37 3 R210L 1, 4 10, 8A & H 37 3 L212I 1, 1 10, 8 A & H 37 3 N213E 1, 1 10, 8 A & H 37 3 N213K1, 2 10, 8 A & H 37 3 D215G 1, 1 10, 8 A & H 37 3 D215W 1, 4 10, 8 A & H37 3 D215C 2, 1 10, 8 A & H 37 3 R226K 1, 1 10, 8 A & H 37 3 N229C 2, 110, 8 A & H 37 3 N229H 1, 4 10, 8 A & H 37 3 N229W 1, 3 10, 8 A & H 37 3A235T 2, 3 10, 8 A & H 37 3 A235C 1, 1 10, 8 A & H 37 3 Q243K 1, 3 10, 8A & H 37 3 Q243R 1, 4 10, 8 A & H 37 3 Q243H 1, 1 10, 8 A & H 37 3 Q243A1, 5 10, 8 A & H 37 3 Q243T 1, 2 10, 8 A & H 37 3 Q243S 1, 2 10, 8 A & H37 3 Q243L 1, 7 10, 8 A & H 37 3 R244V 1, 8 10, 8 A & H 37 3 K254T 1, 010, 8 A & H 37 3 K254H 1, 2 10, 8 A & H 37 3 K254F 1, 6 10, 8 A & H 37 3K254W 1, 7 10, 8 A & H 37 3 G257Q 1, 2 10, 8 A & H 37 3 G257D 1, 6 10, 8A & H 37 3 G257L 1, 7 10, 8 A & H 37 3 G257S 1, 2 10, 8 A & H 37 3 G257M1, 5 10, 8 A & H 37 3 W260Y 1, 6 10, 8 A & H 37 3 W260F 1, 3 10, 8 A & H37 3 N267C 1, 1 10, 8 A & H 37 3 N267Y 2, 4 10, 8 A & H 37 3 N267Q 2, 010, 8 A & H 37 3 N267F 1, 2 10, 8 A & H 37 3 N267E 1, 3 10, 8 A & H 37 3N267M 1, 2 10, 8 A & H 37 3 N267V 1, 1 10, 8 A & H 37 3 A270E 1, 4 10, 8A & H 37 3 A270P 1, 1 10, 8 A & H 37 3 N273Y 1, 7 10, 8 A & H 37 3 N273K1, 3 10, 8 A & H 37 3 N273C 2, 4 10, 8 A & H 37 3 N273Q 1, 3 10, 8 A & H37 3 N273F 1, 2 10, 8 A & H 37 3 N273D 1, 8 10, 8 A & H 37 3 N273S 1, 810, 8 A & H 37 3 A276T 1, 0 10, 8 A & H 37 3 A276P 1, 1 10, 8 A & H 37 3A276D 1, 3 10, 8 A & H 37 3 A276W 1, 3 10, 8 A & H 37 3 N279Q 1, 1 10, 8Model O 40 16  N279E 1, 3 10, 8 A & H 37 3 N279D 1, 2 10, 8 A & H 37 3N279H 1, 2 10, 8 A & H 37 3 N279Q 1, 0 10, 8 A & H 37 3 N283Y 1, 4 10, 8A & H 37 3 N283C 1, 3 10, 8 A & H 37 3 N283F 1, 1 10, 8 A & H 37 3 N283H1, 0 10, 8 A & H 37 3 P285D 1, 0 10, 8 A & H 37 3 P285S 1, 1 10, 8 A & H37 3 P285R 1, 1 10, 8 A & H 37 3 P285K 1, 0 10, 8 A & H 37 3 Y286N 1, 210, 8 A & H 37 3 R289D 1, 1 10, 8 A & H 37 3 R289Q 1, 3 10, 8 A & H 37 3R289L 1, 2 10, 8 A & H 37 3 R289G 1, 0 10, 8 A & H 37 3 R289C 1, 1 10, 8A & H 37 3 R289E 1, 3 10, 8 A & H 37 3 E290D 1, 5 10, 8 A & H 37 3 E290Q1, 0 10, 8 A & H 37 3 S292A 1, 0 10, 8 A & H 37 3 R293T 1, 0 10, 8 A & H37 3 R293E 1, 1 10, 8 A & H 37 3 R293H 1, 1 10, 8 A & H 37 3 R293S 1, 210, 8 A & H 37 3 L294R 1, 1 10, 8 A & H 37 3 L294K 1, 2 10, 8 A & H 37 3L294V 1, 1 10, 8 A & H 37 3 T296S 1, 0 10, 8 A & H 37 3 G301R 1, 1 10, 8A & H 37 3

The residual activity was measured by using the Mannanase enzyme assayusing insoluble Azo-carob-galactomannan substrate from Megazyme.Substrate was incubated with the variants or control for 20 min at 25°C., shaking at 800 rpm. The reaction mixture was kept static for 10 minto allow insoluble substrate to settle. Enzyme activity was measured byreading the optical density of supernatant at 590 nm. The residualactivity was calculated by taking the ratio of Stress response toUn-stress response and expressing in terms of % RA.

Example 4 Thermo-Stability Determination

The variants generated in Example 1 and 2 were screened forthermos-stability at pH 9.0. Stability test was performed by incubatingthe variants in detergent for 105 min at 60° C. and comparing theactivity of variants with a control plate which is incubated at 4° C.for the same duration as described in Example 3. The residual activitywas measured by using the Mannanase enzyme assay using insolubleAzo-carob-galactomannan substrate from Megazyme. Substrate was incubatedwith enzyme for 20 min at 25° C. for 20 min, shaking at 800 rpm. Thereaction mixture is kept static for 10 min to allow insoluble substrateto settle. Enzyme activity is measured by reading the optical density ofsupernatant at 590 nm. The residual activity was calculated by takingthe ratio of Stress response to Un-stress response and expressing interms of % RA.

TABLE 4 Thermo-stable variants of the present invention with improvedhalf life Improvement Factor (HIF) compared to the parent mannanaseSubstitution in Substitution in parent mannanase parent mannanase (SEQID NO: 2) HIF (SEQ ID NO: 2) HIF A1H 1, 1 D118A 1, 0 A1V 1, 1 E133Q 1, 4A1C 1, 1 E133I 1, 0 A1W 1, 1 E133V 1, 2 A1Q 1, 1 D135H 1, 0 A1R 1, 1D135S 1, 3 N2E 1, 4 D135M 1, 1 N2G 1, 2 D135G 1, 2 N2D 1, 1 D135E 2, 3N2W 1, 2 D135Q 2, 2 N2H 1, 3 D135K 3, 1 N2K 1, 0 D135R 1, 5 N2L 1, 3D135V 1, 5 N2Q 1, 3 D135C 1, 1 N2R 1, 2 D135F 1, 1 N2M 1, 3 A136P 1, 4N2T 1, 2 D139P 1, 1 N2F 1, 0 D139R 1, 0 N2S 1, 1 Q143I 1, 1 N2C 1, 2Q143L 1, 1 N2Y 1, 0 P146D 1, 1 N2A 1, 3 R147W 1, 8 S3N 1, 1 R147V 4, 0S3K 1, 2 R147T 2, 4 S3W 1, 4 R147K 1, 2 S3C 1, 1 G152F 1, 2 S3E 1, 2G152A 1, 3 S3Q 1, 2 N154V 1, 2 S3M 1, 1 N154F 1, 0 S3Y 1, 1 N154D 1, 1S3V 1, 1 N154W 1, 1 S3D 1, 1 N154Q 1, 0 S3R 1, 3 W164G 1, 1 S3T 1, 0W164N 1, 8 S8A 1, 1 W164Q 1, 1 S8M 1, 1 W164F 1, 3 S8I 1, 1 W164A 1, 2S8F 1, 2 W164M 1, 4 S8E 1, 2 W164S 1, 0 S8V 1, 2 W164H 1, 1 S8K 1, 0F167Y 1, 2 S8L 1, 2 H172W 1, 7 S8Q 1, 1 H172E 1, 3 S8G 1, 0 R176W 1, 1S8D 1, 2 R176G 1, 5 S8W 1, 1 R176K 1, 3 G9A 1, 1 R176Y 1, 1 G9S 1, 7R176I 1, 2 G9N 1, 4 N180Q 1, 1 G9K 1, 3 A181V 1, 2 G9R 1, 2 A181S 1, 1T11D 1, 2 A181C 1, 2 Y13F 1, 1 A181E 1, 1 Y13E 1, 2 A181Q 1, 1 A15R 1, 0A181R 1, 1 N18F 1, 0 P183T 1, 3 N18H 1, 1 P183I 1, 1 P19V 1, 0 P183N 1,3 P19S 1, 0 P183C 1, 5 P19T 1, 1 P183M 1, 2 D34N 1, 0 P183D 1, 3 D34E 1,4 P183S 1, 2 D34S 1, 3 P183K 1, 4 D340 1, 0 P183A 1, 3 D34T 1, 2 P183E1, 0 Q35V 1, 3 P183H 1, 1 T37A 1, 1 P183W 1, 5 T37Q 1, 3 Q184M 1, 3 E41A1, 2 Q184F 1, 0 E41I 1, 0 R185C 1, 1 E41H 1, 2 N200T 1, 5 E41C 1, 0S202M 1, 2 E41T 1, 2 S202H 1, 1 E41S 1, 2 S202E 1, 3 E41W 1, 1 S202F 1,0 N45K 1, 0 S202D 1, 0 N45R 1, 3 S202N 1, 2 G47T 1, 1 S2020 1, 2 Q60C 3,5 S202R 1, 1 T62D 1, 3 S202V 1, 0 T62E 1, 4 S202Y 1, 9 T62C 1, 1 Q203A1, 2 T62I 1, 3 Q203L 1, 3 T62Q 1, 0 Q203M 1, 1 T62L 1, 0 Q203V 1, 2 T62V1, 8 T206D 1, 1 K63Y 2, 3 T206C 1, 0 I66R 3, 2 R210M 1, 1 H67F 1, 1R210G 1, 6 T68D 3, 6 R210K 1, 1 R70M 1, 1 R210L 1, 0 R70E 1, 0 N213D 1,0 N71K 1, 1 N213I 1, 1 N71T 1, 0 N213V 1, 0 S74K 1, 0 N213E 1, 2 S74R 1,3 D215N 1, 1 S74H 1, 1 D215C 1, 4 D78P 1, 1 N229C 1, 0 D78Q 1, 0 A235T4, 6 D78S 1, 0 A235C 1, 8 D78L 1, 1 Q243L 1, 7 H80W 3, 3 Q243T 1, 5 H80D1, 2 Q243F 1, 5 H80F 4, 9 Q243V 1, 1 H80C 1, 0 Q243E 1, 6 H80S 2, 2R244V 1, 8 Y93M 1, 4 R244K 1, 3 Y93L 1, 1 K254T 1, 1 Y93K 1, 0 K254F 1,4 Y93D 1, 9 K254H 1, 6 Y93E 1, 2 K254W 1, 3 Y93H 2, 1 G257Q 1, 3 Y93Q 1,4 G257D 1, 2 Y93C 1, 7 G257W 1, 7 Y93S 1, 5 G257F 4, 3 S95E 1, 5 G257L1, 5 S95T 1, 1 G257A 1, 2 S95D 3, 2 G257S 1, 0 S95C 1, 7 G257H 1, 3 A97E2, 0 G257M 1, 4 A97C 1, 3 W260L 2, 2 A97D 1, 1 W260L 1, 9 N100M 1, 3W260Y 1, 1 R110T 1, 3 W260F 1, 6 R110S 1, 2 E261A 1, 3 R110C 1, 2 N267C1, 5 R110H 1, 3 N267Y 3, 7 R110G 1, 0 N267R 1, 0 R110K 1, 4 N267Q 1, 1R1100 1, 1 N267F 1, 1 R110V 1, 0 N267E 1, 5 R110A 1, 5 N267H 1, 6 R110L1, 1 N267W 1, 0 R110Y 1, 2 N267M 1, 2 S111N 1, 0 N267V 1, 5 S111A 1, 0N267L 1, 5 S111T 1, 3 N272T 1, 0 S111D 1, 1 N273Y 1, 3 I1140 1, 1 N273H1, 5 I114K 1, 0 N273C 1, 6 I114N 1, 0 N273Q 1, 0 I1141 1, 0 N273F 1, 2I114V 1, 2 N273S 1, 5 G115A 1, 1 A2760 1, 2 G115F 1, 1 A276D 1, 2 G115K1, 0 N279E 1, 2 G115L 1, 1 N279H 1, 0 G115N 1, 1 N283Y 1, 2 G115T 1, 1N283W 1, 0 N283H 1, 4 R293K 1, 1 R293H 1, 0 T296P 1, 1 12965 1, 2 1299E1, 1 T299C 1, 2 T299S 1, 0 G300M 1, 1 G300D 1, 2

Example 5 Detergent Stability Determination

Variants generated as described in Example 1, were screened fordetergent stability at pH 9.0 in Model O detergent for 16 hrs at 40° C.Stability test was performed by incubating the variants in detergent asdisclosed in Table 6 (see below) for 16 hrs at 40° C. (pH 9.0) andcomparing the activity of variants with control plate which wasincubated at 4° C. for the same duration.

TABLE 5 Model detergent O Content of compound Compound (% w/w) Na-LAS(linear alkylbenzene sulphonate) 5.3 AEOS (Alkylethoxy sulphate) 10.7Soyfatty acid 1.0 AEO (Alcohol polyethoxylated) 5.3 TEA (Trietanolamine)0.4 Sodium citrate 2.0 CaCl₂ 0.02 Water 75.3 Water hardness adjusted to12° dH by addition of CaCl₂, MgCl₂, and NaHCO₃ (Ca²⁺:Mg²⁺:HCO³⁻ =2:1:4.5) to the test system. After washing the textiles were flushed intap water and dried.

TABLE 6 Variants having improved half life Improvement Factor (HIF)compared to the parent mannanase when determined at pH 9.0 Mutation HIFMutation HIF N2H 1, 1 N180E 1, 1 N2C 1, 1 N180M 1, 1 N2Y 1, 1 N180Y 1, 0Y6S 1, 3 N180S 1, 2 Y6V 1, 2 N180T 1, 0 Y6K 1, 3 A181V 1, 2 Y6F 1, 8A181I 1, 0 Y6L 1, 2 A181E 1, 0 Y6A 1, 1 A181K 1, 1 Y6R 1, 3 A181H 1, 4S8Q 1, 0 P183D 1, 1 S8G 1, 1 P183S 1, 1 G9S 1, 1 Q184F 1, 2 G9K 1, 1R185C 1, 3 Y13F 1, 1 R185E 1, 2 Y13L 1, 0 R185Y 1, 1 A15R 1, 0 R185K 1,1 G17H 1, 2 N200S 1, 7 R23I 1, 8 A201E 1, 3 K33P 1, 2 S2021 1, 1 D34A 1,0 S202F 1, 0 D34E 1, 8 S202Q 1, 4 D34S 1, 1 Q203A 1, 2 D34T 1, 3 R205I1, 2 D34I 1, 6 R205V 1, 1 Q35H 1, 1 T206I 1, 4 Q35M 1, 0 T206P 1, 0 Q35C1, 1 T206D 1, 0 T37C 1, 0 T206M 1, 1 T37H 1, 2 D215F 1, 0 T37L 1, 0D215N 1, 2 T37S 1, 4 D215L 1, 1 T37N 1, 1 T228M 1, 0 E41Q 1, 0 N229Y 1,3 A44N 1, 0 A235E 1, 1 A44R 1, 0 E242Y 1, 3 N45K 1, 1 G245K 1, 3 G59E 1,0 G257H 1, 3 G59W 1, 0 P258F 1, 2 G59F 1, 1 E261D 1, 3 Q60N 1, 1 E261A1, 1 Q60C 2, 4 N267K 1, 2 Q60M 1, 0 N267D 1, 2 T62F 1, 2 N267W 1, 1 T62H1, 0 A270Y 1, 2 K63L 1, 1 A270M 1, 0 I66H 1, 3 A270C 1, 1 I66R 4, 7A270Q 1, 0 H67R 1, 0 G271I 1, 1 H67N 1, 0 N272K 1, 0 H670 1, 0 N272E 1,1 H67F 1, 2 N273W 1, 0 H67G 1, 1 N273A 1, 1 T68R 1, 0 N273V 1, 1 T68D 2,2 N273M 1, 1 R70G 1, 2 A276Q 1, 0 R70N 1, 1 Y286K 1, 1 N71W 1, 1 L294T1, 1 N71A 1, 0 T299E 1, 1 S74W 1, 1 T299C 1, 0 E77F 1, 0 T299S 1, 1 E77Y1, 5 T299Y 1, 1 D78K 1, 1 T299H 1, 3 D78T 1, 1 T299T 1, 1 D78Y 1, 0T299Q 1, 0 D78M 1, 1 T299M 1, 1 D78N 1, 3 T299L 1, 2 N79H 1, 2 T299G 1,0 N79C 1, 0 G300S 1, 3 N79K 1, 3 G300V 1, 0 H80W 2, 0 G300M 1, 0 H80D 1,0 G300A 1, 1 Y93E 1, 6 G300P 1, 2 Y93H 1, 1 G300F 1, 0 S95C 1, 2 G300L1, 2 I96F 1, 0 G300Q 1, 0 A97N 1, 1 G300D 1, 1 A97V 1, 2 G301E 1, 3 A97M1, 1 G301F 1, 3 N100D 1, 2 G301H 1, 0 N100M 1, 6 G301D 1, 1 N100C 1, 1G301A 1, 2 N100K 1, 0 G301W 1, 1 D104M 1, 0 G301I 1, 1 D104I 1, 0 G301Q1, 2 D104H 1, 0 G301C 1, 1 E108Q 1, 1 G301L 1, 3 E108T 1, 0 G301V 1, 1S111H 1, 0 D135E 1, 5 S111Y 1, 1 D135Q 1, 4 S111V 1, 1 D135K 1, 5 I114A1, 1 D135R 1, 5 I114E 1, 2 D135V 1, 6 I114G 1, 2 D135C 1, 3 I114L 1, 1D139C 1, 0 I114R 1, 2 Q143C 1, 3 I114T 1, 0 Q143E 1, 4 I114V 1, 0 Q143N1, 0 G115H 1, 0 Q143H 1, 1 G115T 1, 0 Q143T 1, 2 K116C 1, 0 Q143V 1, 3K116T 1, 2 Q143W 1, 5 K116S 1, 1 P146D 1, 2 K116N 1, 1 R147E 1, 1 K116V1, 0 N150D 1, 1 D118H 1, 2 N150A 1, 2 D118L 1, 3 N150E 1, 0 D118N 1, 1H172W 1, 3 D118Q 1, 1 H172F 1, 3 D118S 1, 2 H172E 1, 4 E133C 1, 0 D173E1, 2 E133L 1, 1 D173C 1, 0 D135N 1, 2 Y174M 1, 0 D135G 1, 2 R176H 1, 1E177M 1, 0 R176I 1, 0 E177W 1, 1 E177C 1, 1

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated mannanase variant, wherein said variant has mannanaseactivity and comprises a modification at one position corresponding to aposition selected from the positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13,14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44,45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80,81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115,116, 118, 119, 131, 132, 133, 135, 136, 139, 142, 143, 146, 147, 150,152, 154, 164, 167, 169, 172, 173, 174, 175, 176, 177, 180, 181, 183,184, 185, 196, 199, 200, 201, 202, 203, 205, 206, 210, 212, 213, 214,215, 226, 228, 229, 230, 234, 235, 241, 242, 243, 244, 245, 250, 254,257, 258, 259, 260, 261, 262, 266, 267, 268, 270, 271, 272, 273, 276,279, 280, 283, 285, 286, 288, 289, 290, 292, 293, 294, 295, 296, 298,299, 300, and 301 of the polypeptide of SEQ ID NO: 2, wherein eachmodification is independently a substitution or a deletion, wherein saidvariant has at least 65%, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the mature polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ IDNO:
 2. 2. The variant of claim 1, wherein said alteration is asubstitution, wherein said substitution of the naturally-occurring aminoacid residue at the one position for a different amino acid residueproduces a mannanase variant having an Improvement Factor of ≥1.0 for ameasure of stability.
 3. The variant of claim 1, wherein said alterationis a deletion, wherein said deletion of the naturally-occurring aminoacid residue at the one position produces a mannanase variant having anImprovement Factor of ≥1.0 for a measure of stability.
 4. The variant ofclaim 1, wherein said improved stability is in-detergent stability orthermostability.
 5. The variant of claim 1, wherein said variantcomprises a substitution or deletion at one position corresponding topositions 1, 2, 3, 5, 6, 8, 9, 14, 15, 18, 23, 35, 37, 38, 39, 41, 45,47, 59, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95,96, 97, 98, 100, 104, 107, 111, 114, 116, 118, 119, 133, 136, 139, 142,143, 146, 150, 169, 173, 174, 175, 177, 180, 183, 184, 185, 199, 201,202, 203, 205, 206, 210, 212, 213, 214, 230, 234, 235, 241, 242, 243,244, 245, 257, 258, 259, 260, 261, 271, 273, 276, 279, 280, 283, 286,288, 294, 295, 299, and 300 of the polypeptide of SEQ ID NO: 2, andwherein said substitution or deletion of the naturally-occurring aminoacid residue at the one position produces a mannanase variant having anImprovement Factor ≥1.0 for a measure of stability at pH 9.0.
 6. Thevariant of claim 1, wherein said variant comprises a substitution ordeletion at one position corresponding to positions 1, 2, 3, 8, 9, 10,13, 16, 17, 18, 19, 21, 35, 37, 38, 41, 44, 45, 47, 59, 60, 62, 67, 70,71, 74, 77, 78, 80, 93, 95, 97, 100, 107, 110, 111, 115, 118, 132, 133,135, 136, 139, 142, 143, 147, 150, 152, 154, 164, 167, 169, 174, 176,177, 180, 181, 183, 184, 185, 200, 202, 203, 205, 206, 210, 212, 213,215, 226, 229, 235, 243, 244, 254, 257, 260, 267, 270, 273, 276, 279,283, 285, 286, 289, 290, 292, 293, 294, 296, and 301 of the polypeptideof SEQ ID NO: 2, and wherein said substitution or deletion of thenaturally-occurring amino acid residue at the one position produces amannanase variant having an Improvement Factor ≥1.0 for a measure ofstability at pH 10.8.
 7. The variant of claim 1, wherein saidImprovement Factor is at least 1.0 for a measure of stability at pH 9.0and/or pH 10.8.
 8. The variant of claim 1, wherein said variantcomprises a substitution or deletion at one position corresponding topositions 2, 3, 5, 8, 14, 16, 17, 18, 35, 37, 38, 41, 45, 47, 62, 70,71, 77, 78, 80, 81, 82, 83, 93, 95, 97, 98, 100, 107, 110, 116, 132,133, 135, 136, 150, 152, 164, 169, 176, 177, 180, 183, 184, 185, 202,203, 205, 206, 210, 215, 229, 234, 235, 242, 243, 244, 254, 257, 258,260, 261, 267, 273, 276, 279, 283, 288, 290, 294, and 295, wherein saidsubstitution or deletion of the naturally-occurring amino acid residueat the one position produces a mannanase variant having an ImprovementFactor ≥1.5 for a measure of stability at pH 9.0 and/or pH 10.8.
 9. Thevariant of claim 1, wherein said variant comprises a substitution ordeletion at one position corresponding to positions 3, 5, 14, 16, 18,38, 41, 45, 47, 62, 71, 77, 78, 80, 81, 93, 95, 97, 98, 107, 110, 133,135, 150, 164, 169, 176, 177, 183, 184, 185, 202, 205, 215, 229, 243,235, 267, 273, 288, 294, and 295 of the polypeptide of SEQ ID NO: 2, andwherein said substitution or deletion of the naturally-occurring aminoacid residue at the one position produces a mannanase variant having anImprovement Factor of ≥2.0 for a measure of stability at pH 9.0 and/orpH 10.8.
 10. The variant of claim 1, wherein the different amino acidresidue is selected from the group consisting of A, C, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W, and Y, with the proviso that thedifferent amino acid residue is different from the naturally-occurringamino acid residue.
 11. (canceled)
 12. (canceled)
 13. (canceled) 14.(canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled)19. A composition comprising a variant according to claim
 1. 20. Thecomposition according to claim 19, wherein said composition furthercomprises an additional enzyme, such as a protease.
 21. The compositionof claim 19, wherein said composition further comprises a surfactant, ableaching system, a chelating agents, stabilizing agents, hydrotopes,builders, co-builders, bleach activators, polymers and/or fabric-huingagents.
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled) 26.(canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)31. An isolated polynucleotide encoding the variant of claim
 1. 32. Anucleic acid construct comprising the polynucleotide according to claim31.
 33. An expression vector comprising the polynucleotide according toclaim
 31. 34. A host cell comprising the polynucleotide according toclaim
 31. 35. A method of producing a mannanase variant, comprising: a.cultivating the host cell according to claim 34 under conditionssuitable for expression of said variant; and b. recovering said variant.36. (canceled)
 37. A method of dishwashing in an automatic dishwashingmachine, comprising the steps of adding said composition of claim 19 ina detergent composition compartment in said automatic dishwashingmachine, and releasing said composition during a main-wash cycle.
 38. Amethod of laundering in an automatic laundering machine, comprising thesteps of adding said composition of claim 19 in a detergent compositioncompartment in said automatic laundering machine, and releasing saidcomposition during a main wash cycle.
 39. (canceled)
 40. (canceled) 41.(canceled)